CA1138332A - Preferential signal production on a surface in immunoassays - Google Patents

Abstract

PREFERENTIAL SIGNAL PRODUCTION
ON A TEST STRIP IN IMMUNOASSAYS

ABSTRACT OF THE DISCLOSURE
An assay method and compositions are provided for determining the presence of an analyte in a sample. The analyte is a member of an immunological pair (mip) of immunogens--ligand and receptor. The method has two basic elements: a solid surface to which one of the members of the immunological pair is bonded and a signal producing system, which includes a catalytic member bonded to a mip, which signal producing system results in a measurable signal on said solid surface related to the amount of analyte in the medium. The signal generating compound is produced without separation of the catalyst labeled mip bound to the solid surface from the catalyst labeled mip free in solution.
In a preferred embodiment, an enzyme is bonded to a mip which acts in conjunction with a solute to produce a signal generating product which binds preferentially to the solid surface when the enzyme is bound to the surface, resulting in-a signal which is readily differentiated from signal generating compound produced by the catalyst and solute in the bulk solution.

Description

~3~

~ rhere is continuing interest in d~veloping new, -simpler and more rapid techniques t~ méasllre the presence of an analyte in a sa:mple su~pected ~ of containing an analyte .
The analyte may be any of a wide ~ariety of materials, such as drugs, naturally occurring physiological compo~mcls, pol~
lutant~, fine chemicals, contaminants, or the like. In many cases, ~peed is important for the measurement, particularly with certain physiologically active compounds. In other situations, convenience can be a major consideration.
One convenient techni~ue which has found wide application i~ the use of a "dip ~tick." ~aving a solid rod or film which can be dipped in a sample and ~hen sub~Pquently processed to produce a siynal based on the ~amount of a~alyte in the original sample can provide many conveniences. There is ample instrumentation to measure a signal, ~uch as l:ight absorptio~ or fluorescence, ~f a compound bound to a solid ~urface. Al~o, the dip sticlc allows for convenient handling, transfers/ 6eparations, and the like.
In developing an assay, it is desirable that ~here ~e a minimum nu~ber of steps and transfers in performing the ~ssay, as well as a minimum numbe~ of separate .reagents.
Therefore, while a dip ~tick add~ a convenience to ~epara-~ions, the separation~ in themselves are unde~irable. Fur-~ermore / the fewer the reagents that have to be pacXaged, 30 added, and formulated, the fewer the errors which will be introduced into the assay and the grea ter economies and con~renience of the as~ay .

.
. . ..

:. ' ~ - :

`3~

It is therefore desirable to develop new assay methods, particularly employing rigid solid surfaces which may ox may not be separated from the assay medium for mea-surement, where the signal may be develop~d without concern as to the presence o reagents in the solution affecting the observed signal on the solid surfaceO

Patents concerned with various immobiliY.ed reagents in different types of test strips include U.S. Patent Nos.
3,993,451; 4,038,485; 4,046,514; 4,129j417; 4,133,639; and 4,16Q,008, and Ger. Offen. 2,636,244. Patènts disclosing a variety of methods involving separations of bound and unbound antigen include U.S. Patent Nos. Re. 29,169j 3,9497064;
3,984,533;. 3,985,867; 4,020,151; 4,~9,652; 4,067,959 ;
4,108,972; 4,145,406, and 4,168,14~ . `
( Patents of particular interest) A method is provided employing a relatively rigid insoluble, preferably bibulous, surface to which is ~onjugat-ed a member of an immunological pair.(abbreviated as "mip")the immunological pair consisting of ligand and a receptor which specifically binds to khe ligand or their functional equivalent for the purposes of this invention. .In addition to the surface, a signal producing system is provided which has as one member a catalyst, norm~lly an enæyme, which is conjuyated to a mip~ Depending upon the amount of analyte present, the c~talyst labeled mip will be partitioned between the bulk solution of the assay medium and the surface. The signal producing system provides a signal generating compound at the surface which generates a signal which is not signifi-cantly affected by any signal generating compound produced or present in the bulk solution. Therefore, the signal qenerat-ing compound may be generated in the assay medium in the presence o~ unbound catalyst labeled mip. When the only catalyst in the ~i~nal producing system is khe catalyst-labeled-mip, ~ar.ious expedienks can be employed to enhance the difference in ~he rate of formation of the signal gener-ating compound at the surface as compared to the bulk solu . :.
,: ..

. , . :

933~

tion, e.g. enhancing the catalyst turnover rate at the surface. In addition to enhance the simplicity of this protocol, the last of the components of the signal generating system will be added at about the time of or before the addition of the catalyst bound to the mip.
Compositions are provided for performlng the assay comprising combinations of the surface and various reagents in relative amounts ~or optimizing the sensitivity and accu-racy of the assay.
The subject assay provides for a convenient method for detecting and measuring a wide variety of analytes in a si~ple, efficient, reproducible manner, w~ich can employ visual inspection or conventional e~uipment for measuring a spectrophotometric propert~ of a product bound to a sur~ace.
In accordance with the subject invention, an assay method and compositions are provided for measuring a wide variety of analytes, where the analyte is a member of an immunological pair (mip), the pair consisting of a ligand and a receptor (antiligand) which specifically binds to the ligand,or their functional equivalent for the purposes of the assay. The assay method has two e~sential elements: a surface to which is conjugated a mip; and a ~ignal producing system which results in a si~nal generating compound associ-ated with the surface, producing a detectible signal-in an ~nount related to the amount of analyte in the assay medium.
Preferably, the signal producing system will effect a two or more step conversion involving one or more compounds to pxoduce, block or de~troy the signal generating compound, where the rate of change in the concentration of the signal generating compound is related to the avPrage distance between two molecules on the surface. The molecules may be the same or different. The immunological binding at the surface allows for localized enhanced concentrations of compounds o the ~ignal producing system at the suxface.
Also, one may employ a scavenger as a third component which acts to inhibit the operation at the signal producing system in the bulk solution by scavenging an intermediate, catalyst or signal generating compound in the bulk solution.
`' ' The surface may be any convenient structure which substantially retains its form and may ~e separable from or part of the container. The manner of binding o.f the mip to the surface is not a critical aspect of this invention, so long as a sufficien-t amount of the mip is exposed to allow for binding to its homologous partner.
The signal producing system has at leas-t two mem~
bers: A catalyst, normally an enzyme, conjugated to a mip;
and a solute which underyoes a reaction with a substance bound to thè surface, and thereby directly or indirectly enhances or inhibits the production of a detectible signal.
The association of a member of the -signal producing system with the surface may be as a result of insolubilization, complexation with a compound on the surface or interaction, including reaction, with a compound on the surface.
Where an intermediate material is produced by the signal producing system in sol~le form, both in the bulk solution and at the surface, a scavenger can advantageously be employed, so as to substantially minimize the interaction of the intermediate material produced in ~he bulk solution with the surface.
A wide variety of different systems may be employed for altering the degree of production of the product at the surface as compared to the bulk solution and fox inhibiting intermediates or product produced in or migratiny into the bulk solution from interacting with the surface. Depending upon the particular pxotocols, various additions, incubation steps, and reagents will be employed.
By providiny for the production of a detectible signal generatin~ material on the surface that is related to the amount of analy~e in a sample, one can relate the signal level detected from the surface to the amount of analyte in the solution. By employing standards having known amounts of analyte under the same ox substantially the same conditions as with an unknown, one can quantitate the detected signal level with the amount of analyte in the sample.
In accordance with the subject invention, the method is performed wi~hout regu.iring a separation of bound , and unbound catalyst-bound mip, nor requiring a separation of analyte from the remainder of the sample, although the latter may be desirable. This provides substantial advantages in the convenience of the protocol and in avoiding the diffi~
culties in achieving a clean separation.
The subject invention achieves a precise, specific and sensitive techni~ue for detecting and measuring ligands and ligand receptors. The method provides for the preferen-tial production, i~hibition of production or destruction of a compound at a rigid surface, which compound is involved with the generation of a signal at the surface. The signal gener-ating compound associ~ted with the surface will be of a sufficient depth on or in the surface to provide a measurable signal.
For a large number of analytes, the concentration range of interest will fall between 100~g to one pg per ml.
For many analytes, the concentration range of interest will vary from about two-fold to 100-fold so that a quantitative determination will require the ability to distinguish small ~0 differences in the concen-tration of the analyte in the assay medium. Immunoas~ays are predicated on detecting the com-plexation between ligand and receptor, where one or both may be labeled. The lower the concentration of the analyte, the fewer the number of complexes which are formed. Therefore, in order to be able to accurately-determine the number of labeled complexes which are formed, either the label must provide a siynal which can be efficiently counted at an extremely low level of events, e.g. radioactive emission, or the complex must permit amplification or multiplication, e.g.
fluores~ence ox a catalyzed reaction.
When employing ~n amplification system, many prob-lems are encountered. One serious problem is signal result-ing from other than labeled complexes, namely background.
Background signal can result from materials in ~he sample;
labeled contaminants when labeling the member of -~he immuno-logical pair, and un~ound labeled member. In developing an assay, the signal generated by labeled complexes must not be obscured by the signal from the background and must be sub-~3~3~

stantially greater than the ~ackground signal. Therefore any amplification achieved by the signal generating system must be primarily, if not solely, associated wi~h the labeled comple~ rather than with background label.
In many assay techni~ues a clean separation of labeled immune complex and bac~ground label is re~uired, where careful attention must be given to non-specific ef-fects~ For example, where a fluorescent label is employed in a heterogeneous system, e.g. dipstick, after combining all of the reagents with the dipstick, the dipstick must be removed and carefully washed to remove any fluorescer which is non~
specifically bound. Furthermore, the number of fluorescers involved with a complex is limited to the nu~ber which can be conveniently conjugated to a member of an immunolo~ical pair, although further amplification can be obtained by employing a second labeled receptox which binds to a first receptor which binds to a ligand analyte. This step re~uires an additional reagent, another addition and a careful separation to avoid non-specific interastions.
The subject invention obviates or minimizes many of the shortsomings of other methods. For each complex a plu-rality of signal generating events are achieved by employing a catalyst. The catalyst is partitioned between ~he bulk solution and a surface i~ proportion to the ~nount of analyte in the assay medium. The production of signal generating product resulting from the catalyzed reaction at the surface is substantially independent of concurrent production of signal generating pro~uct, if any, produced in the bulk solution. Thus, the assay operates with the catalyst present in the bulk solution during the time the modulation of the amount of signal generating compound at the surface is occur-ring. The need for separating the surface from the bulk solution, whether careful or not, for measuring the si~nal is avoided in the subject invention, although the separation may be preferable.
Furthermore, in the subject inv ntion, ~he siynal generating compound can ~e of suhstantial depth on or in the surface. The presence of the catalyst at the surface allows 3~33~:

for the deposition ox conversion of a large number of signal generating compounds to provide a strong sic~al. This is of great importance when the measurement is vi~:ual inspection, particularly where the signal generation involves the absorp-tion o light.
Before ~urther describing the invention, a number of terms will be defined.
DEFINITIONS
Analyte - the compound or composit:ion to be mea sured, which may be a ligand, which is mono or polyepitopic, usually antigenic or haptenic, a single or plurality of compounds which share at least one common epitopic or deter-minant site or a recep~or.
Specific binding pair - two diferent molecules,' where one of the molecules has an area on the su'rface or in a cavity which specifically binds to a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as ligand and receptor (antiliyand~. These will be referred ~o in the subject application as members of an immunological pair, abbreviated as "mip". ~omologous or complementary mips are ligand and receptor, while analogous mips are either ligands or recep-tors, which are differentiated in some mar~er, e.g. labeling.
Li~and ~ any organic compound for which a receptor naturally exists or can be prepared~
Receptor (antiligand) - any compound or composition capable of recognizing a particular spatial and polar organ-ization of a molecule i.e. epitopic or determinant site.
Illustrative receptors include naturally occurring receptors, e.g. -thyroxine binding globulin, antibodies, enzymes, Fab fragments, lectins, nucleic acids and -the like.
Ligand Analog - a modified ligand which can compete wi~h the analogous ligand for a receptor, the modification providing means to join a ligand analog to another molecule.
The ligand analog will usually differ from the ligand by more than replacement of a hydrogen with a bond which links ~he ligand analog to 2 hub or label, but need not.

: . :.

~f~3~3~

Polytligand-analog3 - a plurality of ligands or ligand analogs covalently joined together, nQrmally to a hub nucleus. The hub nucleus is a polyfunctional material, normally polymeric, usually having a plurality o~ functional S groups e.g. hydroxy, amino, mercapto, ethylenic, etc. as sites for linking. The hub nucleus is normally water soluble or at least dispersible and will usually be at least about 35,000 daltons, but generally not exceeding about 600,000 daltons. Illustrative hub nuclei include polysaccharides, polypeptides, including proteins, nucleic acids, ion e~change resins and the like.
Surface - the surface will-be non dispersed ancl of a dimension of at least about l~m2 and generally greater, often at least about ln~2, frequently ~from about O.Scm2 to lOcm2, usually being on a support when less than about 0.5cm2; and may be of any material which is insoluble in water and provides the necessary properties for binding of a mip and a detectible signal generating compound ~o provide a desired signal level. Desirably, the surface will be gelatinous, permeable, porous or have a rough or irregular structure, which may include channels or indentation~, gener~
ally having a substantial void volume as compared to total volume. Dependin~ upon the nature of the detectible signal generating compound, the surface will be adsorbent or non-adsorbent, preferably being weakly or non-adsorbent. The surface may be transparent or opaque, a single material or a plurality of materials, mixtures or laminates. A wide var-iety of materials and shapes may be employed. The surface will be capable of substantially retaining its integrity under the conditions of ~he assay so that substances which are ~ound to th~ surface will remain bound to the surface and not diffuse into solution.
Signal producing ~ystem - the signal producing system has at least two members: (1) a cakalytic member; and 3S ~2) a solute, which undergoes a reaction cataly~d by the catalytic member, which leads directly or indirectly to a product on or in ~he surface which provides a detectible signal. Desirably, a third compound will be present which .

` ~3~`3~

provides for enhanced rate of change of the signal generatlng compound at the surface as compared to the bulk solution.
This can be as a result of the component be:ing bound to the surface or interactin~ with another member of the signal producing system.
The catalytic member may be enzymatic or non-enzymatic, preferably enzymatic. Whether one or more than one enzyme is employed, there will be at least one enzyme bound to a mip. ~An enzyme acting as a catalyst should be distinguished from an enzyme acting as a receptor.) The solute can be any compound which is capable of und~rgoing a reaction catalyzed by a catalytic member of the signal producing system, which reaction results either directly or indirectly in modulating the formation of a detectible signal generating compound associated with the surface. The association of the signal generating compound to the surface may be as a result of insolubilization of the product produced when solute undergoes the catalyzed reac-tion, complexativn of the product with a compound on the surface or reaction or interaction of a compound on the surface with the product of the catalyzed reaction.
The signal generating ~ompound will provide an electromagnetic signal, e g. a spectrophotometric or visible, electrochemical or electronic detectible signal. The signal generating compound will be ~ssociated with the surface due to its insolubility, or covalent or non-covalent binding to the surface. The observed detectible signal rom the surface will be related to the amount of catalyst bound to the sur-face through the binding o the catalyst-bound-mips to the mip-bound-surface.
Various technigues and combinations of reagents may be employed to enhance the production of the detectible signal at the surface, while minimizing intererence from materials in the bulk solution.
Label - the label may be any molecule conjugated to another molecule where each of the molecules has had or can have had 2 prior discrete existence. For the most part, labels will be compound~ conjugated to a mip. In referring ,, . ............ .. .. ..
..

to a catalyst conjugated to an antiligand, the reagent will be referred to as a cataly~t~bound-antiligand, while for a ligand conjugated to a surface, the reagent will be referred to as ligand-bound surface.
Method _ The subject assay is carried out in an aqueous zone or medium, where the final assay medium may be the result of prior individual additions of reagents or combinations of reagents and incubations, prior separations involving removal of the surface from an aqueous medium and transfer to a different aqueous medium having one or more reagents, or combinations thereof. The subject method, however, does not require a separation of catalyst-bound-mip which is unbound from that which is bound to its homologoùs partner bound to the surface (mip~-bound-surface). The medium consists of a liquid phase and a non-fluid phase which is the "surface."
In carrying out the assay, the mip-bound surface will be contacted ~y the sample, and by the members of the signal producing system, and any ancillary materials in an aqueous medium, either concurrently or stepwise, to provide a detectible signal associated with the surface. The detecti-ble signal will be related to the amount of the catalyst-bound-mip bound to the surface, which in turn will be related to the amoun~ of analyte in thè sample. Depénding upon the nature of the signal produ~ing system and the desired method for detec-ti.ng the ~ignal, the surface may be xead in the assay medium or will be read separate from the assay medium.
In carrying out the assay, an aqueous medium will normally be employed. Other polar solvPnts may also be included, usually oxyyenated organic solvents of from 1-6, more usually from 1-4 carbon atoms, including alcohols, ethers and the like. Usually these cosolvents will be present in less than about 40 weight percent, moxe usually in less than about 20 weight percent.
The pH for the medium will usually be in the range of about ~-11, more u~ually in the range of about 5-10, and preferably in the range of about 6.5-9.5. The pH i~ chosen so as to maintain a significant level of specific binding by ~3~3;~

the receptor while optimizing signal producing efficiency.
In some instances, a compromise will be made between these two considerations. Various buffers may be used to achieve the desired pH and maintain the pH during the determination.
Illustrative bufEers include borate, phosphate, carbonate/
Tris, barbital and the like. The particular buffer employed is not critic~l to this invention but in individual assays, one buffer may be preferred over another.
Moderate tempera-tures are normally employed for carrying out the assay. Constant temperatures durin~ ~he period of the measurement are generally required only if the assay is performed without com~arison with a control sample.
The temperatures for the determination will generally range from about 10-50~C, more usually from about 15-45C.
The concentration of analyte whlch may be assayed will generally vary from about 10 4 to 10 15M, more usually from about 10 6 to 10 13M. Considerations such as whether the assay is qualitative, semi quantitative or guantitative, the particular detection technique and the concentration of t.he analyte of interest will normally determine the concen-tration of the other reagents.
The concentrations of the various reagents will vary widely depending upon which protocols are employed, the nature of the analyte, the mip which is bound to the surface and the mip which is bolmd to the catalyst, the required sensitivity of the assay, and the like. In some instances, large excesses o one or the o~ler of the mips may be em-ployed, while in some protocols the sensitivity of the assay will be responsive to variations in the mip ratios.
By way of illustration, if the analyte is a poly-epitopic antigen, one could have excesses of antiligand as antiligand-bound~surface and as catalyst-bound-antiligand, without seriously affecting the sensitivity of the assay, provided that the surface is first contacted by the sample, followed by contact with the ~ignal producing system. Where antiligand is the sample and the protocol involves the com-bination of the analyte and cataly~t-bound-antiligand prior to contacting ~he antigen-bound-surface, the sensitivity of .

.

~3~

the assay will be related to the ratios of ~he analyte and catalyst bound~antiligand concentration.
In addition to the considerations involving the protocol, the concen-tration of the reagents will depend on the binding constant of the antiligand, the binding constant profile for a particular antisera, as well elS the required sensitivity of the assay. Also, when all 0.1-- the signal producing system is present in the liquid phase, the catalyst substrates and ancillary reagents should be at a concentra~
tion which allows for substantial immunological pair binding befcre a large amount of signal producing product is formed.
Where the sensitivity of the assay is concentration related, fre~uently the particular concentrations wilI be determined empirically. When the sample i6 combined with the homologous catalyst bound-mip, generally the total binding site concen-tration of the catalyst-bound-mip will be not less than abou-t 0.1 times the minimum concentration of interest based on binding sites of analyte and usually not more than ahout 1,000 times the maximum concentration of interest based on analyte ~inding sites, usually about 0.1 to 100 times, more usually about 0.3-10 times the maximum concentration of interest. When the analyte is preadsorbed to the mip-bound-surface, the concentration of catalyst-bound-mip will depend on the desired rate of ~inding to the surface, the production of interfering signal generating compound in the liguid phase, the cost of the reagent, etc.
The concentration of catalyst-bound mip will be chosen so that the amount of catalyst~bound~mip in the void ~olume-llquid immediately adjacent to and occluded in the surface will not significantly interfere with the measurement of the change in concentration of the signal generating compound at the sur~ace as a result of catalyst~bound-mip bound to the surface. The chosen concentration will be affected by the sensitivity of the measurement, the degree of quantitation desired, the accuracy with which one must dis-tinguish the lowest concentration of interest and the like.
In most situations, the ratio of concentration in the void volume of catalyst bound-mip unbound to the surface ~3~3~

to catalyst-bound-mip bound to the surface should be not greater than about 100 fold, usually not greater than about 10 fold at the ma~imum concentration of interest of the analyte, preferably at the mid-range concentration range of int~rest of the analyte.
The combination of the solid surface with the sample may be prior to, concomitant with, Ol. subsequent to combining the catalyst-bound-mip with the sample. By employ~
ing a single unit or entity as the sllrface, one can use the surface to concentrate the analyte in a large sample. Also, the surface allows for removal of the analyte from other materials in the sample which could interfere with the deter~
mination of the result. Therefore, a preferred embodiment will be to con~ine the surface with the sample, followed by removal of the surface from the sample containing medium and transfer to the assay medium.
Alternatively, one could leave the surface in contact with the sample and add the remaining reagents. It is also feasible, although in some instances no-t desirable, to combine the sample with the catalyst-bound-mip, followed by introduction of the surface into the assay medium. For example, with a ligand analyte, en2yme-bound-antiligand and ligand-bound-~urface, this last technigu could be effective-ly used.
Fre~uently, the last of ~he components of the signal producing system will be added at about the same time as the catalyst-bound-mip, without any intermediate step, such as separating or washing the sur~ace.
Where a receptor is the analyte, instead of having a single immunological pair, one may employ two immunological pairs, where the receptor acts as the ligand in one pair and the receptor in the o~her. For example, with IgE, one could bind the allergen or antigen to the surface and bind the catalyst to anti-IgE. In this way, the IgE acts as a bridge between two mips which in themselves cannot interact. In referring to a mip, this situation ~hould be considered a ~pecial case which is intended to be included.

, . :;.,, ' :

~3~3!`3~

1~
In developing protocols for the method, certain basic considerations will govern -the order of addition and the combinations of reagents. The first consideration is that preferably where the surface-bound-mip and the catalyst-bound mip are different members e.g. one is ligand and one isantiligand, the two will be brought together prior to or substantially concomitant with combination w.ith the surface.
The catalyst-bound-mip and solute will preferably be combined as a single reagent, e~cept when the ~olute i~ the substrate of the catalyst-bound-mip. Frequently, the surface and sample will be combined prior or nearly concomitant with the addition of the other xeagents.
Various protocols`will have various degrees of complexity. In the simpler protocols, there will be two catalysts involved in the signal producing system, one which is bo~md to a mip, and the other bound to the surface. One catalyst, pr0ferably the surface-bound-catalyst, reacts with the solute to pxoduce a first product. This first product is acted on by the second catalyst, which first product by itself or in combination with other reagent~ produces a second product which preferen-tially binds to the surface or interacts with a compound bound to the surface, when produced adjacent to the surface. This can be achieved conveniently by producing a second product which i~ insoluble. By insolu-ble is intended a solubility of less than about 10 3M. Theinsoluble product may effect changes in electrical properties e.g. electrostatic or have spectrophotometric prcperties, including absorption in -the ultraviolet or visible wavelength range, chemiluminescence, reflectance and fluorescence, preferably absorption.
In order to minimize the amount of repetition, a table is provided which assembles various illustrative pro-tocols. While the table is directed to polyepitopic anti-gens, haptens can be employed in place o the antigens.
However, with haptens it will norrnally not be convenient to bridge between receptors, ~nd in protocols that require bridging, the addition of a poly~ligand analog) is required to provide the bridging. When the analyte is a hapten, one ~3~

will normally add the hapten containing sample to the xeceptor. When the catalyst bound-mip is ~he receptor, the mip bound to the 6urface is normally hapten. When the mip bound to the surface is a receptor, the mip bound to the catalyst is normally hapten. Thus, one will normally satur-ate a portion of the receptor binding sites with the hapten analyte and cause the remaining s.ites to combine with the hapten either conjugated to the surface or to the catalyst.
The antigen or polyepitopic analyte a~ a ligand offers additional flexibility in that the xeceptor may be bonded to both the catalyst and surface, without addeA
poly(ligand analog). Where the llgand is bonded to the surface, the ligand analyte and thè ligand on the surface may compete for a limited amount of labeled receptor or a polyvalent receptor act as a bridge between ligand-bound-surface and catalyst-bound-ligand. Where receptor is bound to the surface, the ligand may then act as a bridge binding simultaneously to the receptor-bound-surface and catalyst-bound-receptor, ~o as to bind catalyst-bound-receptor to the surface. In the latter situation, where the receptor-bound-surface and ligand containing sample are combined prior to addition of ~he catalyst-bound-receptor, one can have a large excess of labeled receptor since the ~mount of labeled recep-tor which ~inds to the surface will be directly related to the amount of ligand bound to the surface. Where receptor-bound-surface is employed with a receptor analyte, the two receptors may complete for a limited amount of catalyst-bound-antigen.
Where ligand is the analyte and ligand is bound to the surface, one will normally first combine the sample containing ligand with catalyst-bound receptor, so that the li~and and catalyst-bound-receptor may bind and caus~ a reduction in the number of available sites of ~he receptor that can be scavenged by the ligand bound to ~he surface.
Where receptor is bound to the surface, and ligand is the analyte, any order of mixing will be operable, although it would usually be desirable to combine the sample with the surface~ before co~tacting the surface wi~h the catalyst~

.
, :

3~

bound-receptor. Normally, the formation of siynal generating compound will be followed as a rate, observing the change in signal on the surface with time. The rate of course will be related to the amount of label which binds -I;o the ~urface.
This measurement may be made prior to establishing full equilibrium between the analyte, catalyst-bound--mip and mip-bound-surface, and thus the rate may vary with time.

TABLE I
Protocols .
_ _ Materlals Added1 _ Surface Conjugated Anc.

2 mip ~e Sample Solute Reagent fAg ~-Catl Ag ~ +
II 1 Ab-Cat1 Ag 2fAg +
III 1 fAb Ab-Catl Ag + +
IV 1fP~D Ag Ab-Catl +
V 1Cat2fAg Ab-Catl Ag ~ +
20 VI 1 Ab-Catl Ag + +
2cat2f~g VII 1 Cat2~Ab Ab-Catl Ag ~ +
VIII 1 Cat2fAb Ag 2 Ab-Catl ~ +
25 IX 1RfAb Ab-C~tl Ag + +
X 1RfAb Ag 2 Ab-Catl ~ -~
XI 1 Ab-Catl Ag 2RfAb + -~
30 XIII 1 fAg Ab-Catl ~b + +
XIV 1 RfAg Ab-Catl ~b ~ -~
XV 1 Cat27~g Ab~Catl Ab +

Surface mip - member of an immunological pair bound to a surface in addition to other members of the si~nal producing sys-tem. The f s~mbolizes the surface. ~g and ~3~3;2 Ab mean ligand and antiligand respectively, bound covalently or non-covalently to the surface, where in any given protocol the roles of ~b and Ag may be reversed.
Cat2 - means a catalyst, usually an enzyme, which cooperates with another catalyst usually an enzyme as members of the signal producing system.
R - reagent, bound covalently or non~covalent ly to ~he surface which reacts wilh the product of a catalyst as part of the si~nal producing system.
Conjugated mip - ligand or antiligand to which a catalyst, usually an en~yme, is covalently bonded.
Catl - a catalyst~ usually an enzyme, which is part of the signaI producing system and reacts with the solute or product formed from -the solute.
Solute - a medium soluble compound which reacts with Catl or Cat2 as part of the signal producing system.
Anc. reagents - any additional reagents necessary to the signal producing system, including enzymes, enzyme substrates and cofactors, activators, scavengers and the like.
2Each line indicates that the materials on that line are co~bined prior to the addition of any of the materials on the next line. The materials on each line may be added concur-rently or consecutively, although in many instances one or the othe~ order of addition will be pre~erred. When the surface is combined with the sample prior to addition of the conjugated mip, the surface may or may not be separated from ~he sample prior to contacting the surface with the conju-gated mip and other reagents. Incubation steps may be in-volved between steps and between the addition of materials as part of one step.

Various protocols can be invol~ed by using one or more catalysts in combination with a solute and one or more intermediate members of ~le signal producing system. In developing the protocols, one is concerned with maximizing the production of the signal, so that the signal generating .
' ;

~3~3`~

molecule is preferentially produced at and on the surface.
Furthermore, it i6 desirable that the reagents be combined in as few separate formulations as pos6ible, so as to minimize the number of measurements and additions whlch are reguired.
Where one has a catalyst bound to a mip, which reacts with the solute to produce a signal generator which precipitates within the surface pores or channels, the sur-face need not be separated from the assay medi~ for reading.
I f removed from the assay medium as a matter of convenience in measuring the signal generator on the surface, it need not be washed to remove any non-specifically bound signal genera-tor or catalyst-bound-mip.
One can further enhance the localized production of the signal generator at the surface by having two or more catalysts, particularly.enzymes. By employing as a solute a substrate of one of the enzymes, preferably an enzyme bound to the surface, where the product resulting from the solute is the substrate for another enzyme, normally bound to a mip, one can significantly minimize the rate of production of signal generating compound in ~he bulk solution produced by the enz~me bound to the mip. In effect, by having the sub-strate for the catalyst bound-mip produced at the surace, one can minimize the rate of production of signal generating compound produced in the bulk solution, since the concentra-tion of such substrate in the bulk solution will generally be~uite small.
In addition to haviny substrate produced at the surface, other techniques may be employed to minimize produ~-tion of the signal generating compound in the bulk solution.
For example, in the example given above, one could employ a scavenser in the bulk solution which would act upon the product of the solute~ to prevent its further reaction.
Alternatively, or in addition, one can employ an enzyme inhibitor, which is added after binding of the enzyme-bound-mip to the surface which is effective with the enz~me in thebulk solution, but not effective with enz~me bound to the surface. A further alternative, is to have a reagent which is bound to the surface, which reacts with the enzyme product to produce the signal generating compound.

~ ~3~ Z

Another protocol involves the use of an enzyme-bound-mip which prevents the formation of the signal produc~
ing substance on the surface. ~or example, an enzyme may be bound to the surface which catalyzes the conversion of the solute to an intermediate product. A seconcl enzyme bound to the surface is employed to convert this intermediate product to the signal generating compound. The enz~e-bound~mip employs ano~hex enzyme that can react with the intermediate product w.ithout forming the 6ignal gPnerating compound. When the enzyme-bound mip becomes bound to the surace it inhibits the ormation of the signal generating compound on the sur-face. This pxotocol provides the advantage that a minimum signal is produced when the catalyst-bound-mip is maximally bound to the surface. Thus for certain protocols in which the analyte and the catalyst-bound-mip compete for mi~-bound-surface binding sites, the absence of analyte gives a minimum signal and the presence of analyte gives an incroased signal.
In accordance with the above protocols, a signal generating compound is produced or destroyed at the surface 20 iIl xelation to the amount of analyte in a sample. The signal generating compound which is bound to the surface will be substantially unrelated to the amount of signal generating compound, if any, produced in the bulk solution. That is, to the extent that a signal generating compound i~ produced in the bulk solution, the amount which may diffuse from the bulk solution to the surface and bé bound to the surface will be negligibly small compared to the amount of signal gQnerating compound produced at the solid surface at the minimum signal level for ~he concentration xange of interest of the analyte.
The choice in bindins the ligand or receptor to the surface will depend on a number of factors. When a polyepi-topic ligand is the analyte, the use of catalyst~bound- ^-receptor enhances the assay response by permittin~ many catalysts to become bound to each molecule of the analyte that binds to the surface. The purity of the li~and or receptor will also be of significance. Since anti~era are frequently heterogeneou~ and may have only a small proportion of the desired receptox, the use of catalyst-bound-receptor ' may produce excessive amounts of signal generating compound in the bulk solution. One should therefore compare the purity of the ligand to the receptor in determining to which mip the catalyst should be conjugated. Furthermore, since in 5 many situations the concentration of the analyte of interest will be extremely low, the binding of -the members of the mip may be relatively slow. Therefore, if one can use a large excess of the mip on the surface homologous to the analyte, the rate of binding of the analyte and the :resultant develop-ment of the signal generating compound at the ~urface can begreatly enhanced.
Another consideration is the convenience and effi-ciency of combining the maximum number of rea~ents in the fewest number of formulations. By emp1oying a system with two or more catalysts, one can combine an enzyme catalyst with the substrate of another en~,yme, referred to as the solute. In addition, one can also combine any ancillary reagents necessary for the two catalysts in a single reagent, since the catalytic r~action of the enzyme cannot occur until the other enzyme produces its substrate.
As is evident, the orders of addition and combina-tion of the various reagents, including the introduction of the surface into the assay medium can be varied widely.
Where there is a compekition for a limited number of binding sites, either of the ligand or the receptor, normally the sample will be initially bound to its homologous mip ~ coun-terpart) prior to the addition of a competitive analogous mip. Or, all of the reag~nts may be combined simultaneously.
The other reagents necessary for producing the signal gener-ating compound may then be added concurrently with the analo-gous mip or subsequent to combining the analogous mip. In addition, it will be desirable, particularly where the signal producing system employs a single catalyst, that the rate of formation of the signal generating compound in the assay medium be affected by a component which differentiates between the surface and the bulk solution. Factors such as the control of local change, pH, solute concentrations, etc.
on the surface can be employed to produce differential enæyme actlvity.

3Q~

Frequently, when one is combining the sample with its homologous mip bound to the surface, an incubation step will be involved, to allow for a s~stantial c~mount of the analyte to bind. A second incubation step may be involved where the catalyst-bound-analogous mip combines with the remaining bindlng sikes of the homologous mip-bound-surface or where the ligand acts as a bridge for two receptors, one conjugated to ~he surface and the other conjugated to the catalyst label. Whether a second incubation step is involved will depend to a substantial degree on the rate of binding, the sensitivity re~uired for the assay, and the rate of production of signal generating compound at the solid sur-face. Conveniently, one combines the catalyst-bound-mip and remaining members of the signal producing system substantial-ly concurrently and allows the signal generating compound tobe produced while the catalyst-bound-mip is bindin~ to the surface.
The followiny are illustrative of a few exemplary protocols. In the first exemplary protocol, a single enzyme catalyst is employed, which is bound to a receptor e.g.
antibody. A porous surface is employed to which is also bound receptor. The sample containing polyepitopic li~and analyte i~ combined with the antibody-bound-surface and the mixture incubated for a sufficient time, so ~hat a detectable amount of analyte would have had an opportunity to bind. To the mixture is then added the enzyme-bound antiligand and the mixture incubated again for a sufficient time for a detect-able amount of the enzyme conjugate to bind to ligand bound to the surface. Buffer may be included with the enzyme-bound~antiligand to enhance the binding of the enzyme conju-gate to the ligand.
After sufficlent incubation, the solùte and any other reagents fox measuring enzyme activity may be intro-duced as a single reagent, including an agent which enhances

3~ the enzyme activity at the surface as compared to the bulk solution, for example, a macro-molecular enzyme inhibitor e.g. polyantienzyme. The i~hibitor would be sterically precluded from binding to enz~me bound to the surface.

~L3~

Alternatively, one or more or all of the necessary substrates or cofactors may be combined with the enzyme-bound-antiligand and introduced with khe enzyme-bound-antiligand into the assay medium. So long as an essential component for the S enzyme reaction is withheld, the other reagents necessary for the enzyme reaction may be included with the enzyme as a single reagent. After adding the necessary reagents for the enzyme reaction, one can wait a sufficient time period for the signal generating compound to be produced within the porous surface and compare the signal thus produced to a reference signal, e.g. signal produced with a known amount of analyte. Alternatively, one could take two readings and determine the change in intensity of the signal with time.
Another pQssibility is after a predetermined time from the complete addition of all o the substrates and cofactoxs necessary for the enzyme, the surface is removed and read outside of the assay medium.
As distinct from using the ligand as a bridge, a hapten analyte is illustrative of a competition mode. In this exemplary protocol, the sample containing the hapten analyte would be combined with enzyme-bound-receptor and the hapten-bound-surface to which is bonded a precursor to the ~ignal generating compound and, as appropriate, the mixture incubated for a sufficient time for the haptén to bind to the receptor and the enzyme-bound-receptor to the ~urf~ce. The solute and ancillary reagents are then added to the assay medium where the enz~me produces a product which reacts with the precursor to produce the signal generating compound. One could then take one or more readings at predetermined time intervals to determine the rate at which the ~ignal generat~
ing compound is produced on the solid surface, which would be related to the number of available binding sites of ~he receptor after binding of the hapten analyte in the sample.
Another alternative with a single enzyme is to employ an oligomeric substrate with an exohydrolase. For example, one could have a disaccharide bonded to a dye which provides ~ colorless reagent, which on removal of the sugar provide~ an insoluble colored dye. Slnce the di~accharide ~31~

requires two en~matically catalyzed hydrolyses, in effect one has a third component since the one enzyme acts on two different substrates. There is, therefore, an analogous situation to the two enzyme system, where the action of one enzyme in a first stage produces as a product a substrate for a second en~yme, which acts in a second stage. With such a substrate, the inclusion of an agent that enhances the enz~me a~ti~ity at the sur~ace as compared to the bulk solution is of less importance.
In a third exemplary protocol, employing two enzymatic catalystsl both an enzyme and antibody would be bound to the surface ( en7yme and antibody bound-surface).
The surface would be combined with a polyepit~pic antigen analyte and the mixture inoubated for a sufficient time for the antigen to bind to the receptor on the surface. Normal-ly, the binding of the antigen will be performed in the undiluted sample. To the mixture may then be added as a single reagent the enzyme catalyst bound receptor, the solute, which is the substrate for the enzyme bound to the surface, and the remaining reagents/ including any additional precursors to the signal generating compound where the pro-duct resulting from the solute i~ not the only precursor to the signal generating compound. Alternatively, the surface can be transferred to this single reagent. As previously indicated/ the signal may be read on the surface-in ~he assay medium/ or the surface may be removed from the assay medium and read elsewhere. The signal which is detectable from the surface will be proportional to the amount of catalyst-bound-mip bound to the surface, which in turn is proportional to the amount of analyte in the sample.
In~tead of having a second enzyme on the surface, one could bind two different enzymes on different mips, either both on ligands or both on receptors, or bcth enzymes on the same mip. The significant factor is that the immuno-logical pair binding results in the enhanced localized concentration of two members or molecules of the signal produc-ing system at the ~urface, which members interact to enhance the change in concentration of the signal generating com~
pound, wi~hout reacting with each other.

3~

The employment of a two stage process for modula tion of the signal generating compound may or may not involve a third reagent in addition to the solute and catalyst-bound-mip as a component of the signal producing e;ystem. The significant factor is that the immunological binding at the surface provides an opportunity for concentrating components of the signal producing system at the surface as compared to the bulk solution.
Where the cooperation or interaction of the compo-nents of the signal producing system in the overall rate ofproduction or destruction of the signal generating compound is related to the average spatial proximity of the components of the signal producing system, the binding of the catàlyst-boun~-mip through immunological pair ~inding to the surface permits enhancement of the locallzed concentration of compo-nents of the ~ig~al producing sy~tem as compared to the bulk solution, so as to minimize the effect of any generation of signal generating compound in the bulk solution on the amount of signal generating compound at the ~urface.
Alternatively or in addition, a scavenger can be added which preferentially reacts or interacts with a compo-nent of the signal producing system other than the solute in the bulk solution. The scavenger acts to interfere with the operation of the signal producing system in the bulk solution by either preventing a catalyzed or non-catalyzed reaction from proceeding or preventing a signal generating compound from generating a signal.
Normally, the sisnal will be by observation of electromagnetic radiation, particularly ultraviolet or visi ble light, either absorption or emission, particularly ab sorption, or electrical properties of the surface. Desir-abl~, light will be in the range from about 250 to 800nm, usually fxom about 350 to 700nm. Visual inspection~ reflec-tometers, fluorometers, spectrophotometers or the like may be employed, depending upon the signal ~enerating compound and the nature of the surface, that is, whether opaqu2 or krans-parent. Usually, it will be the intensity (transmission or emission~ of the signal yenerator on the s~lrface which will be correlated with the amount of analyte.

.

3~

The temperature at which the signal is observed will generally range from about -lso to 50C, more usually from about 15 to 40C.
5tandard s~mples can be prepared which ~ave known amounts of analyte. The observed signal for each of the standard samples may then be plotted or compared visually, so as to relate concentration to signal. Alternatively, a number of surfaces may be prepared relating to various con-centrations, and visual or spectroscopic comparison made between the surface of the sample and the standards. Depend-ing upon the accuracy re~uired, the ~tandards may be made as a prior color chàrt or may be made ~y the analyst determining the sample. Once a standard curve has been established, an observed signal may be directly related to the concentration of the analyte.
In a preferred method for calibration, a surface is employed that is identical to the surface employed in the assay but without a mip bound to it. For the assay of a polyepitopic anti~en using receptor-bound-surface and catalyst~bound-receptor, the failure of catalyst-bound-receptor to bind to the surface indicates that no antigen is present in the sample. Since the calibration surace cannot bind catalyst-bound-receptor even in the presence of anti~en, the surface provldes a suitable comparison for ~egative samples when subjected to the iclentical protocol as the mip-bound surface employed with the sample. By comparing the signal from the calibration surface with the signal from ~he mip-bound-surface, any difference is indicative of the presence of antigen.
Another alternative .is to employ a calibration ~ystem involving mips different from the analy~e and its homologous mip. Conveniently, one would modify the catalyst or catalyst bound-mip with a hapten recognized by a receptor bound to the calibration surface or employ a receptor for a natural site on the catalyst or catalyst-bound-mip unrelated to the specific mip binding.
By adjustment of the concentration of the receptor on the calib~ation surface a simulated assay response can be !
' ', ' ~

:' :
. ~ .

~3~

produced that is identical to ~he signal produced by a pre~
determined (usually zero3 concentration of analy~e.
As stated above, the calibration surface and the assay surface (mip-bound-surface) are subjected to identical assay conditions and the signals compared. Depending upon whether an increase or decrease in signal results Erom an elevation in analyte concentration, a difference in signal between the calibration surface and the ass~y sur~ace in the appopriate direction would indicate the presence of analyte.
The time for measuring the signal will be basecl on such factors as the sensitivity re~uired, concentration of analyte, rate o~ bindi~g, nature of the signal producing system, etc. Since at zero time there is no chan~e in the initial signal, a single measurement need only be made at the end of the final incubation. For better quantitation, mea-surements could be made at intervals during the incubation, with incubation time varying from 5secs to 36hrs.
The ligand analyte may be mono- or polyepitopic.
Except ~hat a hapten cannot be employed as a brldge between the receptor bound to the ~urfac~ and catalyst-bound-receptor, hapten and antigen analytes can be treated analo-gously. If a bridge is desired, a poly(ligand analog) may be employed having a plurality of haptens joined together and employiIlg a limited amount of catalyst-bound-receptor. In this protocol, the receptor-bound-surface would be co~bined with the sample and poly(ligand analog), followed by addition of catalyst-bound-receptor. Of course, one could replace the poly(ligand analog~ and catalyst-bound-receptor, by hapten conjugated to the catalyst (hapten-bound-catalyst).
Where the receptor is the analyte, one can allow for competition between the receptor analyte ahd receptor-bound surface for a limited amount of catalyst-bound-ligand.
For a receptor analyte, alternatively, one could employ the antigen as a bridge as described pr viously, where the recep-tor analyte competes with catalyst~bound-receptor for the antigen bound to the receptor as recep-tor-bound-surface.
In ~he event that the analyte, the mip bound surface, and the catalyst bound-mip are all the same member, ~3~

then the homologous member must be added and in polyepitopic form either, for example, for receptor, as an anti.body or a polyvalent receptor, where the receptor is other ~than an antibody, or or ligand, as polyhapten (poly(ligand analog)) or polyepitopic an-tigen.
The subject method lends itself to the determina-tion of a plurality, two or more, analytes simultaneously.
By having a surface e.g. a strip, with a plurality of mips for different analytes, e.g. antigens, so that each mip is localized at a particular position on the solid surface, in combination with a plurality o~ catalyst-bound-mips, specific for each analyte, generation of sIgnal at each site would be indicative of different analytes.
Materials : .
The components employed in the subject assay will be the analyte; the surface; the signal producing system; and as appropriate poly(ligand analog) or polyvalent receptor.
The signal producing system will have at least two members, the catalyst-bound-mip and the solute, frequently having additional members.
Analyte The ligand analytes of this invention are charac-terized by being monoepitopic or polyepitopic. The poly-epitopic ligand analytes will normally be poly~amino acids) i.e. polypeptides and proteins, ~olysaccharides, nucleic acids, and combinations thereof. Such combinations of assem blages include bacteria, viruses, chromosomes, genes, mitochondria, nuclei, cell membranes, and ~he like.
For the most part, the polyepitopic ligand analytes employed in the subject invention will have a molecular weight of at least about 5,000, more usually a:t least about lO,000. In the poly(amino acid) category, the poly(amino acids~ of interest will generally be rom about 5,000 to 5,000,000 molecular weight, more usually from about 20,000 to l,000,000 molecular weight; among the hormones of interest, the molecular weights will usually range ~rom about 5,000 to 60,000 molecular weight.

.' The wide variety of proteins may be considered as to -the family of proteins having similar structural features, proteins having paxticular biological function~, proteins related to specific microorganism.s, particularly disease causing microorganisms, etc. For cells and viruses, histocompatability antigens or surface antigens will fre~
~uently be of interest.
The following are classes of proteins related by s-tructure:
protamines histones albumins globulins scleroproteins phosphoproteins mucoproteins chromoproteins lipoproteins nucleoproteins glycoproteins proteoglycans unclassified proteins, e.g. somatotropin, prolactin, insulin, pepsin A number of proteins found in the human plasma are important clinically and include:
Prealbumin Albumin al-Lipoprotein al-Acid ~lycoprotein al-Antitrypsin al-Glycoprotein Transcortin

4.6S-Postalbumin Tryptophan-poor ~l-glycoprotein alx-Glycoprotein Thyroxin-binding glo~ulin Inter ~-trypsin-inhibitor Gc~globulin ~Gc l-l) (Gc 2~1) (Gc 2-2) ~I~ptoglobin ~Hp l-l) ~Hp 2-1) (~p 2-2) Ceruloplasmin `
Cholinesterase~
a2-Lipoprotein(s) Myoglobin C-Reactive Protein a2-Macroglobulin a~-HS-glycoprotein Zn-~-glycoprotein a2-Neuramino~glycoprotein Erythropoietin ~-lipoprotein Transferrin Hemopexin Fibrinogen Plasminogen ~-glycoprotein I
~2 glycoprotein II
Immunoglobulin G
(IgG) or yG-globulin ~ol. formula:
y2K~ or y2A2 Immunoglobulin A (I~A) or yA-globulin Mol. fo~mula:
(~2K2~ or (a2A2) Immunoglobulin M
(IgM) or yM~globulin :

:, ~ , , . :

.

Mol. formula:
(~2K2)5 ~r ~A~)5 Immunoylobulin D(I~D) or yD-Globulin (yD3 ~ol, formula:
(~2K2) or ~A2 Immunoglobulin E (IgE~
or yE-Globulin (yE) Mol. formula:
(2K2) ~ (2 2~
Free K and A light chains Complement factors:
C'l ~ ' l q C'lr :
C'ls Cl2 C'3 ~lA
~2D
C'4 C'5 C'6 C'7 C'8 C'9 Important blood clotting factors include:

BLOOD CLOTTING FACTORS

30 International designation Name .
I Fibrinogen II Pxothrombin IIa Thrombin III Tissue thromboplastin V and VI Proaccelerin, accelera-tor globulin ~3~

VII Proconvertin VIII Antihemophilic globulin ~AHG3 IX Christmas factor, plasma thromboplastin component ~PTC) X Stuart-Pxower factor, autopro~hro~bin III
XI Plasma thromboplastin antecede:nt (PTA) XII Hagemann factor XIII Fibrin-~tabilizing factor ., , '~

Important protein hormones include:
~etide and Protein Hormones .
Parathyroid hormone (parathromone) Thyrocalcitonin Insulin Glucagon Relaxin Eryt~ropoietin Melanotropin . ~ -(melanocyte-stimulating ~5 hormone; intermedin) Somatotropin (growth hormone) Corticotropin (~drenocorticotroplc hormone) Thyrotropin Follicle-stimulating hormone Luteinizing hormone (interstitial cell-stimulating hormone) 3~ Luteomammotropic hormone (luteotropin, prolaGtin) Gonadotropin (chor1onic gonadotropin3 .. ..
.

; .

3~

Tissue ~ormones _ _ _ Secretin Gastrin Angiotensin I and II
Bradykinin ~uman placental lactogen Peptide Hormones from the Neurohypophysis Oxytocin Vasopressin Releasing factors (RF3 CRF, LRF, TRF, Somatotropin-RF, GRF, FSH RF, PIF, MIF .
Other polymeric materials of interest are mucopolysaccharides and polysaccharides.
Illustrative antigenic polysaccharides derived from microorganisms are as follows:
Species of Microorganisms Hemosensitin Found in Streptococcus pyogenes Polysaccharide Diplococcus pneumoniae Polysaccharide 20 Neisseria meningitidis Polysaccharide Neisseria gonorrheae Polysaccharide Corynebacterium diphtheriae Polysaccharide Actinobacillus mallei; Crude extract Actinobacillus whitemori 25 Francisella tularensis . Lipopolysac-charide Polysaccharide Pasteurella pestis Pasteurella pestis Polysaccharide 30 Pasteurella multocida Capsular antiyen Brucella abortus Crude extract Haemophilus influenzae Polysaccharide Haemophilus pertu~sis Crude Treponema reite A Polysaccharide 35 Veillonella Lipopolysac-charide Erysipelothrix Polysaccharide 1isteria monocytogenes Polysaccharide ' .`~

` ~3~3~

Chromobacterium ~ipopolysac-charide Mycobacterium tuberculosis Saline extract of 90% phenol extracted mycobacteria and polysac-charide fraction of cells and turberculin Klebsiella aerogenes Polysaccharide Klebsiella cloacae Polysaccharide Salmonella typhosa - Lipopolysac-charide, Polysaccharide Salmonella typhi-murium; Polysaccharide Salmonella derby Salmonella pullorum ::
20 Shigella dysenteriae Polysaccharide Shigella flexneri Shigella sonnei . Crude, Poly-saccharide Rickettsiae . - -Crude extract 25 Candida albicans Polysaccharide Entamoeba histolytica Crude extract The microorganisms which are assayed may be intact, lysed, ground or otherwise fragmented, and the resulting composition or portion, e.g. by extraction, assayed. Micro-organisms of interest include:
Corynebacteria Cor~nebacterium diptheriae Pneumococci .
Diplococcus pneumoniae Streptococci Streptococcus pyogenes Streptococcus salivarus , , . ,, , ~

- . - ,. : : ,: : "
- .

.. . ~ ,.
.. . . ~ .

~3~

Staphylococci Staphylococcus aureus Staphylococcus albus ~eisseriae Neisseria meIlingitidis Neisseria gonorrheae Enterobacteriaciae Escherichia coli Aerobacter aerogenes ~ The coliform Klebsiella pneumoniae ¦ bacteria Salmonella typhosa .
. Salmo~ella choleraesuis ~ The Salmonellae Salmonella.typhimurlum J

Shigella dysenteriae Shigella schmitzii Shigella arabinotarda > The Shigellae Shigella flexneri Shigella boydii Shigella Sonnei Other enteric bacilli \

Proteus vulgaris ....... 1 Proteus mirabilis ~ Proteus species Proteus morgani Pseudomonas aeruginosa Alcaligenes ~aecal:is Vibrio cholerae ~emophilus-Bordetella_~rou~
Hemophilus influenzae, H. ducreyi H. hemophilus H. aegypticus ~. parainfluenzae Bordetella pertussis ~3~

Pasteurellae Pasteurella pestis Pasteurella tulareusis Brucella Brucella melitensis Brucella abortus Brucella suis Aerobic Spore-formin~ Bacilli Bacillus anthracis Bacillus subtilis Bacillus megaterium Bacillus cereus Anaerobic Spore~-formlng_Bacilli -Clostridium botulinum - .
Clostridium tetàni Clostridium perfringens Clostridium novyi Clostridium septic~
Clostridium histolyticum Clostridium tertium Clostridium bifermentans Clostridium sporogenes Mycobacteria Mycobacterium tuberculosis hominis ~ 25 Mycobacterium bovis Mycohacterium avium Mycobacterium leprae Mycobacterium paratuberculosis Actinomycetes (fungus-like bacteria) A~tinomyces israelii Actinomyces bovis Actinomyces naeslundii Nocardia asteroides Nocardi~ brasiliensis The Spirochetes Treponema pallîdumSpirillum minus Treponema pertenueStreptobacillus moniliformis . . ~ . ~.

Treponema carateum Borrelia recurrentis Leptospira icte.rohemorrhagiae Leptospira canicola coplasmas Mycoplasma pneumoniae Other patho~ens Listeria monocytogenes Erysipelothrix rhusiopathiae Strep~obacillus moniliformis Donvania granulomatis Bartonella bacilliformis Rickettsiae (bacteria like parasites) , Rickettsia prowazekii Rickettsia mooseri Rickettsia rickettsii Rickettsia conori Rickettsi~ australis Rickettsia sibiricus Rickettsia akari Rickettsia tsutsugamushi Rickettsia burnetii Rickettsia quintana Chlamydia (unclassifiable parasites ~acterial/viral) Chlamydia age.nts Inamin~ uncertain) Fun~i Cryptococcus neoformans Blastomyces dermatidis Histoplasma capsulatum Cocciaioides immitis Paracoccidioides brasi~iensis Candida albicans Aspergillus fumigatus Mucor corymbier (Absidia corymbifera) Rhizopus oryzae Rhizopus arrhizus ~ Phycomycetes Rhlzopus nigricans `
' .

3~

Sporotrich~ schenkii Fonsecaea pedrosoi Fonsecaea compacta Fonsecaea dermatidi.s S Cladosporium carxionii Phialophora verrucosa Aspergillus nidulans Madurella mycetomi Madurella grisea Allescheria boydii Phialosphora jeanselmei Microsporum gypseum Trichophyton mentagrophytes Keratinomyces ajelloi:
Microsporum canis Trichophyton rubrum Microsporum andouini Yiru~es Adenoviruses Herpes Viruses Herpes simplex Varicella ~Chicken pox) Herpes Zoster (Shingles) Virus B
Cytomegalovirus Pox Viruses .
Variola (smallpox) Vaccinia Poxvirus bovis Paravaccinia Molluscum contagiosum Picornaviruses Poliovirus Coxsackievirus 3S Echoviruses Rhinoviruses ;

~3~3~

Influenza (A, B, and C) Parainfluenza (1-4 Mumps Virus Newcastle Disease Virus Measles Virus Rinderpesk Virus Canine Distemper Virus Respiratory Syncytial Virus Rubella Virus Arboviruses Eastern Equine Eucephalitis Virus Western Equine Eucephali~is Virus Sindbis Virus Chikugunya Virus Semliki Forest Virus Mayora Virus St. Louis Encephalitis Virus California Encephalitis Virus Colorado Tick Fever Virus ~ellow Fever Virus Dengue Virus Reoviruses Reovirus T~pes 1-3 2S Hepatitis ~epatitis A Virus Hepatitis B Virus Tumor Viruses Rauscher Leukemia Virus Gross Virus Maloney Leukemia Virus Epstein Barr Virus Other Parasites Related to the Followin~ Diseases Doy Heart Worm (microfilaria) Malaria Sc~istosomiasiæ
Coccidosis Trichinosis 3~

The monoepitopic ligand analytes will generally be from ~bout 100 to 2,000 molecular weight, more usually from 125 to 1,000 molecular weight. The analytes of interest include drugs, metabolites, pesticides, pollutants, and the like. Included among drugs of interest are the alkaloids.
Among the alkaloids are morphine alkaloids, which includes morphine, codeine, heroin, dextromethorphan, their deriva-tives and metabolites; cocaine alkaloids, which includes cocaine and benzoyl ecgonine, their derivatives and metabo-lites; ergot alkaloids, which includes the diethylamide oflysergic acid, steroid alkaloids; iminazoyl alkaloids;
~uinazoline alkaloids, isoquinoline alkaloids, ~uinoline alkaloids; which includes guinine ~nd ~uinidine; diterpene alkaloids; their derivatives and me-tabolites.
The next group of drugs includes steroids, which includes the estrogens, gestogens, androgens, andrenocortical steroi~s, bile acids, cardiotonic glycosides and aglycones, which includes digoxin and digoxigenin, saponins and sapogenins, their derivatives and metabolites. Also included are the steriod mimetic substances, such as diethylstilbestrol.
The next group of drugs is lactams having from 5 to 6 annular members, which include the barbitura~es, e.g.
phenobarbital and secobarbital, diphenylhydantonin, primidone, ethosuximide, and their metabolites.
The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3 carbon atoms, which includes the amphetamines, catecholamines, which includes ephedrine, L-dopa, epinephrine, narceine, papaverine, their metabolites.
The next group of drugs is aminoalkylbenzenes, wi~h alkyl of from 2 to 3 carbon atoms, which includes ephedrine, L-dopa, epinephrine, narceine, papaverine, their metabolites and derivatives.
The next group of drugs is benzheterocyclics which 3X include oxa2epam, chlorpromazine, tegretol, imipramine, their derivatives and metabolites, the heterocyclic rings being azepine~, diazepines and phenothia%ines.

3~

The next group of drugs is purines, which includes theophylline, caffeine, their metabolites and derivatives.
The ne~t group of drugs includes those deriYed from marijuana, which includes cannabinol and tetrahydrocannabinol.
The next group of drugs includes the vitamins such as A, B, e.g. B12, C, D, E and K, folic acid, thiamine.
The next group of drugs is prostaglandins, which differ by khe degree and sites of hydroxylation and unsaturation.
The next group of dirugs is antibiotics, which include penicillin, chloromycetin, actinomycetin, tetracycline, terr~nycin, their metabolites and derivatives.
The next group of drugs is the nucleosides a~id nucleotides, which include ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine withi their appropriate sugar and phosphate substituents.
The next group of drugs is miscellaneous individual drugs which include methadone, meprobamate, serotonin, meperidine, amitriptyline, nortriptyline, lidocaine, procaineamide, acetylprocaineamide, propranolol, griseofulvin, valproic acid, butyrophenones, antihistamines, anticholinergic drugs, such as atropine, thieir metabolites and derivatives.
The ne~t group of compounds is amino acids and small peptides which include polyiodothyronines e.g~
thyroxine, and triiodothyronine, oxytocin, ACTH, angiotensin, met-and leu-enkephalin their met~bolites and derivativesO
Metabolites related to diseased states includie spermine, galactose, phenylpyruvic acid, and porphyrin Type 1.
The next group of drugs is aminoglycosides, such as gentamicin, kanamicin, tobramycin, and amikacin.
Many drugs of interest will involve aralkylamine structures, which may or may not be a part of a heterocyclic structure, e.g. alkaloids, phenobarbitol, dilantin, epinephrine, L-dopa, etc. While there is some similarity in structure, the compounds vary widely as to activity.

~3~

Drugs may also be considered a~ to the primary purpo~e for which they are used. In many situations, it is desirable to monitor a drug for police functions, ~herapeutic dosaye monitoring for drugs used for treatment of asthmatics, epileptics, cardiovascular diseases, hypertension, bacterial or viral infection, gastraintestinal infections, etc. In each case, physiological fluids such a~ blood, serum, saliva, etc. are monitored to ensure that the administered drug is within the therapeutic dosage for the indiv:idual.
Among pesticides of interest are polyhalogenated biphenyls, phosphate esters, thiophosphates, carbamates, polyhalogenated sulfenamide~, thei-r metabol:ites and derivatives.
For receptor analytes,.the molecular weights will generally range from 10,000 to 2x106, more usually from 10,000 to 106. For immunoglobulins, IgA, IgG, IgE and IgM, the molecular weights will generally vary from about 160,000 to about 106. Enzymes will normally range from about 10,000 to 6000,000 in molecular weight. Natural receptors vary widely, generally being at least about 25,000 molecular weight and may be 106 or higher molecular weight, including such materials as avidin, thyroxine binding globulin, thyroxine binding prealbumin, transcortin, etc.
Ligand Analog The ligand analog will differ from the ligand either by replacement of a hydrogen or a functionality with a bond or a linking group which has a functionality for forming a covalent bond to another molecule having an active func-tionality, such as an hydroxyl, amino, aryl, thio, olefin, etc., where the resulting compound differs from the ligand by more than substitution of a hydrogen by the molecule to which it is conjugated. The linking group will normally have from 1-20 atoms other than hydrogen, which are carbon, o~ygen, sulfur, nitrogen, and halogen of atomic number 17-35. The functionalities which are involved include carbonyl, both oxo and non-o~o, active halogen, diazo, mercapto, ethylene, particularly activated e~hylene, amino, and the like. The number of heteroatoms will generally range from about 0-6, ;

~ ~3~33;~

more usually from about 1-6, and preferably from about 1-4.
A description of linking ~roups may be found in U.S. Pate~t No. 3,817,837.
For the most part, the linking groups will be aliphatic, although with diazo groups, aromatic groups are involved. Generally, the linking group is a divalent chain having about 1-10, more usually from about 1-6, atoms in the chain. Oxygen will normally be present as oxo or oxy, bonded to carbon and hydrogen, preferably bonded solely to Garbon, while nitrogen will normally be present as amino, bonded solely to carbon, or amido, while sulfur would be analogous to oxygen.
Common functionalities in forming the covalent bond between the linking group and the molecule to be conjugated are alkylamine, amide, amidine, thioamide, urea, thiourea, guanidine, and diazo.
Linking groups which find particular application with conjugation to polypeptides are those involving carboxlyic acids which may be used in conjunction with diimides, or as mixed anhydrides with carbonate monesters or as active carboxlyic esters e.g. N hydroxy succinimide or p-nitrophenyl.
Nitrogen analogs may be employed as imidoesters. Aldehydes can be used to form imines under reductive amination conditions e.g. in the presence of borohydrides, to produce alkylamines.
Other non-oxo carbonyl groups which may be employed include 2~ isocyanates and isothiocyana-tes. In addition, active halide may be employed, particularly bromoacetyl groups.
In most instances, the ligand will have one or more functional groups which may be employed as the site for linking the linking group. Particularly, hydroxy, amino and aryl groups, particularly activated aryl groups find use.
Also, oximes may be prepared from oxo functionalities and the hydroxyl used as a site for joining to a linking group, such as carboxymethyl.
The choice of linking ~roup will vary widely, depending upon the functionalities which are present in the , ~3~3~

ligand, in the compound to which the ligand is to ~e conju-gated, the nature and length of the linking group desired, and the like.
Solid Surface The surace can be widely varied. Usually, the surface will be chosen so as not to be strongly adsorbent for members of the signal producing system, which would deleter~
iously affect the assay, so as not to interfere with the measurement of the signal generated by the signal generating s~stem and to substantially retains its physical integrity during the assay. The surface may take different forms, have different physical characteristics, can be of ~ifferent chemical compositions and may be of one or more compositions as a mixture of compositions or iamlnates or co~binations thereof. The particular surface will interact with the signal generating compound by desolubilization of the signal generating compound onto the surface, or permi-t complexation, reaction or interaction of a compound bonded to the surface, so as to form or destroy the signal generating compound.
2~ The surface may be of a variety of shapes and forms, as well as of varied dimensions depending on the manner of use and measurement. The surface may be supported by a rod, tube or capillary, fiber, strip, disk, plate, cuvette, or the like. The surface will be an integral pa~t of the support or distinct from the support as an applied layer having a relatively small thickness, usually at least 0.1~, more usually 1~, generally 10~, or greater dependin~ on the nature of the surface, ease of application and desired properties.
The surface may be opaque, translucent or transpar-ent. It may be a solid, gel or viscous liquid, permeable or non-permeable, porous or non porous, bibulous, reticulated7 convoluted, channeled, being uneven or smooth, or coated with a continuous or discontinuous layer. Preferably, the surface will be penetrable by the signal generating compound to at least a depth of 0.1~, more preferably at least 1~ and par~
ticularly preferred at least 10~.

~3~3~2 The surface may also be considered in accordance with its function. The surface serves as a base or substrate which will retain a discrete existence in the aqueous assay solution, ~o as to be discernable from the medium and usually S separable from the medium. The surface serves to support mips which are bound to it, so that -they are incapable of diffusing through the solution independent of the surface.
In addition, -the surface acts as a support for the signal generating compound, either as a base for a deposi-ted lay~r or as a support for covalent or non-covalent attachment. The surface is effectively non-fluid, discrete in ~hat ~he sur~
face is distinguishable from the liquid medium in which the surface is immersed, and provides a distinct base or founda~
tion for supporting mips, members o~ the signal generating system or other compounds as appropriate, which are bound either covalently or non-covalently. The surface may ex.ist in a charged or non-charged form, being charged where such charge provides some advantage to the operation of the signal producing system.
Various materials may be employed, the primary considerations being the binding of the signal generating compound to the surface, the absence of interference of signal generation, the ease of conjugating to the surface, and the like.
A wide variety of organic and inorganic polymers, both natural and synthetic may be employed as the material for the solid surface. Illustrative polymers include poly-ethylene, polypropylene, polyt4-methylbutene3, polystyrene, polymethacrylate, poly~ethylene terephthalate), rayon, nylon, poly(vinyl butyrate), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, etc. Other`materials which may be employed, include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cermets or the like. In addition are included substances that form gels, such as proteins e.g. gelatins, lipopolysaccharides, silicates, agarose, and polyacrylamides or polymers which form several aqueous phases, such as dextrans, polyalkylene glycols (alkylene of 2 to 3 carbon atoms~ or surfactants e.g.

:, 3~

amphiphilic compounds, such as phospholipids, long chain ~12-24 carbon atoms~ alkyl ammonium salts and the like.
Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system. In some instances, the pore si~e may b~ limited, in order to avoid access to catalyst bound to the surface.
Cut off sizes can vary from tens of thousands, e.g. 20,000 to millions dalton, e.g. 20 million, usually cut-off size will not be less than 5000 daltons.
The particular material employed for the solid surface will be insoluble in the assay medium, may be swel-lable or nonswellable, preferably nowswellable, may be hydrophobic or hydrophilic,~i.e. polar or non-polar, prefer-ably hydrophilic, may be coated with a thin mono- or pol~y-molecular layer of a different composition or uncoated, may be a single material or a plurality of materials, particular-ly as laminates or fibers, may be woven, cast, extruded, etched, aggregated, etc.
In preparing the surface, a plurality of different materials may be employed, particularly as laminates, to obtain various properties. For example, a porous layer may be deposited onto a nonporous transparent cuvette wall, which may provide a window for viewing ~he signal, while protecting the adjacent layer. A surface may be modified so as to enhance the hinding characteristics of the signal generating compound, inhibit migration in a particular direction, act as a semi-permeable membrane, or the like. Protein coatings, e.g. gelatin can be employed to avoid non-specific binding, simplify covalent conjugatlon, enhance signal detection or ~he like. However, it should be appreciated that the mater-ial employed should preferably not be strongly adsor~ent, so as to adsorb a ~ignal generating compound which is produced in ~he bulk solution and diffuses to ~he surface.
The particular dimensions of the surface suppcrt will be a matter of convenience, depending upon ~he size of the samples involved, the protocol, the means for measuring the signal, and ~he like.

~ 3~

The surface will usually be polyfunctional or be capcible of being polyfunctionali~ed, so as to allow for covalent bonding between the mip and the surface, as well as the other compounds which must be conjugated. Functional groups which may be present on the surface ~md used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to the various surfaces is well known and is amply illustxated in the literature. See for example Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York, 1978, and Cuatrecasas, J. Biol. Chem 245 3059 ~1970).
The length of the linking group may vary widely depending upon the nature of the compound being linked, the effect of the di.stance between the linked compound and the surface on the linked compound's properties, the potential for cross-linking of the linked compound, and the like.
The linkin~ group may be a bond or have up to about 12, usually not more than about 10 atoms in a chain. The linking group may b~ aliphatic, alicyclic, aromatic, heterocyclic, or combinations thereof. The total number of atoms of the linking group will be not more than about 20, usually not more than about 16 atoms other than hydrogen, which will be carbon, oxygen as oxy or oxo, both o~co-carbonyl ànd non-oxo-carbonyl; nitrogen as amino or amido, and sulfur as thio or thiono. Illustrative groups include methylenecarbonyl, succinimidyl, ~haloacetyl, thiomethylene, glycyl or polyglycyl, succindioyl, maledioyl, glutardlalkylidene, methylenephenyldiazo, and ureido.
A mip will always be bound, covalently or non-covalently to the solid surface. Dependin~ upon the nature of the protocol, the amount of bound mip may be limited or in excess vf the highest amount of analyte which can be expected to be found in the sample based on binding sites of the analyt~. In addition, one or more me~ber~ of the signal producing system may also be bound to the solid surface. Frequently, the amount of ~he member of the signal producing system which is conjugated will be rate limiting, ~o that large excesses will be employed.

-,.
~ .

.
`:

~7 Si~nal Producing System The signal producing system has at least two members: A catalysk, normally an enzyme; and a solute, which is cap~ble of undergoing a catalyzed reaction which may directly provide the signal generating compound, may provide a precur60r to the signal generating compound, or may produce a product which serves to react or interact with another compound to produce, block or destroy the signal generating compo~md .
As already indicated, one or a plurality of catalysts may be employed, but usually at least one of the catalysts will be an enzyme. ~hile there may be two or more catalysts, usually as ~he number -o~f catalys-ts increases over three, the advantages are quickly offset by the disadvan--tages. Thereore, normally there will not ~e more than three catalysts, usually not more than two catalysts.
At least one catalyst will he bound to a mip, either covalently or non-covalently, for example through a specific binding pair. One or a plurality of the same or different catalyst molecules may be bonded to one or more mips or alternatively, a plurality of mips may be bonded to a single catalyst molecule. Where there are a plurality of catalysts, the choice of which catalyst to bond to the solid surface and/or the mip, will depend upon the conveniences involved in formulating the reagents, ease of conjugation and the effect on the sensitivity of the assay. Therefore, there frequently will be a preference as to which catalysts are conjugated to the solid surface and which catalysts are conjugated to the mip.
To provide a siynal preferentially associated with the ~urface as compared to the bulk solution, ~arious tech-niques may be used. These techniques include: insolubiliæa-tion of a signal generating compound; preferential production of a signal generating compound at the surface with binding to the surface; scavenging or inhibition of catalyst-bound-mip in the bulk solution; scavenging of a catalyst, signal ~enerating compound or a precursor in the bulk solution with the component of the signal producing system protected from the scavenger at the suxface; and producing a compound at ~he surface which interacts, including reacts, with a compound on the surface. Not only can these various techni~ues be used individually, but ~hey can also be combined with advantage.
In desolubiliza-tion to provide an insoluble signal generating compound, a compound is catalytic:ally transformed from a soluble form to an in~oluble form; fc,r a dye from a leuco form ~o a colored form; for a fluorescer, from a non-fluorescent compound to a fluorescent compound.
A large number of dyes exist which are predicated on being modified to a colored form as they bind to a variety of natural fabrics. The same type of modification can be involved in the subject inventïon, where the dye can be desolubilized catalytically, particularly enzymatically. The desolubilization may involve oxidationj reduction, or hydrol-ysis, particularly of water solubilizing groups, such as organic and inorganic esters, e.g. phosphates, pyrophos-phates, carboxylates, e.g. uronates, sulfates, and the like, or ethers, such as glycosidyl ethers.
A wide variety of compounds can be modified so as to enhance their hydrophobicity -lack of solubility in an aqueous medium. The compolmds can then be further modified to enhance their hydrophilicity e.g. substitution with water solubilizing substituents which upon catalytic removal results in a product which has gained signal generating capability. For example, phenolic compounds can be esteri-fied with organic or inorganic acids or etherified with sugars. Amines can be acylated. Heterocycles can be oxidized or reduced to enhance solubility or insolubility.
In each instance the reactant will be incapable of signal generation, while the product will be capable of signal generation or influencing signal generation.
Various compounds may be employed as solutes which upon catalytic transformation, either in one or two steps, results in an insoluble electroactive or changed molecule, chromophore or fluorophore product as the signal generator.
In some instance~ a redox reaction may transform a sol~ble compound having light absorption at short wavelengths 3~

to an insoluble compound absorbing light at substantially longer wavelengths. When a commercially available sompound which could otherwise be emp]oyed as a solute does not pro-vide an insoluble product, such commercial:Ly available com-pound could be modified in conven-tional ways by substitution with hydrophobic groups, such as hydrocarbon, e.g. alkyl, halo, e.g. chloro and bromo, cyano, nitro, ox combination~
thereof. Alternatively where the commercially available compound is insoluble, and could fulfill the reguirements of a signal generating compound, such commercially available compound could be employed as a solute if substituted with a catalytically removable group which confers water solub:ility and which effects a substantial change in the electroactive, chromophoric or fluoxophoric properties o the compound.
The subject invention also lends itself to employ colored coupling products employed in photography. By em-ploying a catalyst other than silver, as the catalyst-bound-mip, with a substrate which can be activated to react with a compound bound to the surface to produce a colored coupling product, the signal producing system can parallel color photography. For an excellent xeview and list of composi-tions which may be employed in the subject invetion, see "The Theory of the Photographic Process," 3rd ed. Edited by T. H.
James, The Macmillan Co., New York (1966) pages 383-396.
Of particular interest are such combinations as substituted anilines, particularly .amino-substituted anilines and phenols, particularly naphthols. Individual compounds include a-benzoyl-3-{a-(4-carboxymethylphenoxy)acetamido]-acetanilide; 1-phenyl-3-[3-(4-cyanomethylphenoxy)benzamido~-

5-pyrazalone; 1-[4-(3,5-dimethylphenoxy)phenyl3-3-(4-aminoethoxy-3-methylbenzamido)~5-pyrazalone; and l-hydroxy-N~[y-(2-tert.-amyl-4-carboxymethylphenoxy)propyl]-2~
naphthamide. These compounds can be covalently conjugated to a surface or further modified with lipophilic groups to become non covalently bound to a surface.
The following lists are compounds which may be employed as solutes, which by one or two step catalytic transformations would produce a ~ignal generating compound.
In some instances because of the solubility characteristics of the compounds, some substitution would be necessary to achieve the desired properties. The lis~ oi chromophores and fluorophores is broken down into categories of the nature of the listed reaction.
CHROMOPHORS AND FLUOROPHORE REACl'IONS
__ Redox tetrazolium salt ~ formazan leuco methylene blue ~ methylene blue leuco Meldola blue ~ Meldola blue 4-Cl-l-naphthol -~ colored oxidation product leuco phenazine methosulfate ~ phenazine methosulfate N-3,5-dibromo or N-3,5-dichloro-4 hydroxyphenyl ~-dimethylaminoaniline -~ N~ dimethylaminophenyl) 3,5-dibromo- or 3,5-dichloroquinone monoimine dihydropyocyanine ~ pyocyanine 2-(2'-benzothiazolyl)~5-styryl-3~(4'-phthalhydrazidyl) tetrazolium chloride ~ formazan derivative dihydrosafranine ~ safranine leuco benzyl viologen ~ benzyl viologen diaminobenzidine ~ colored oxidation products o-toluidine -~ colored oxidation product a naphthol ~ pyronine -~ fluorescent product 5 amino-2,3-dihydro 1,4-phthalazinedione ~ hv aminoantipyrene ~ 1,7-hydroxynaphthol -~ colored coupling product ~is umbelliferyl phosphate ~ umbelliferone ~,4-dinitronaphthyl-1 ~-D-galactoside -~ ~,4-dinitronaphthol 3~

N-(2'-methoxyphenyl) 6-bromo~3-carboxamide-naphthyl~
~-D-glucosiduronic acid ether ~ N-~2'~metho~yphenyl)

6~bromo-3-carbo~amide-2 naphthol water soluble The preferred spectrophotometrica].ly active com-pounds - dyes, fluorescers and chemiluminescers- have hydroxyl groups, particularly one or more phenolic groups, which are present in the parent compound or may be int;roduced. The hydroxyl groups are convenient sites for esterification to form e~ters e.g. phosphates and uronate~ or ethers, particu-larly glycosidyl ethers.
As already indicated, both enzymatic and nonenzy-matic catalysts may be employed. Preferably,-enzymatic catalysts will be employed, since they frequently provide for more rapid reactions, a-desirable versatility in the variety of reactions, and have well characterized properties.
In choosing an enzyme, there will be many consid-erations in addition to those involved with the reaction of interest. These considerations include the stability of the enzyme, the desirability of a high turnover rate, the sensi-tivity of the rate to variations in the physical environment, the nature of the substrate~s) and product~s~, the avail-ability of the enzyme, the effect of con~ugation of the enzyme on ~he enzyme's properties, the effect ~n enzyme activity of materials which may be encountered in the sample ~olutions, the molecular weight of the enzyme, and ~he like.
The ollowing are categories of enzymes as set forth in accordance with the classification of the Inter-national Union of Biochemistry.

3~

ABLE II

1. Oxiodoreductases 1.1 Acting on the CH-OH group of dono:rs 1.1.1 With NAD or NADP as acceptor 1.1.2 With a cytochrome as an acceptor 1.1.3 With 2 as acceptor 1.1.99 With other acceptors 1.2 Acting on the aldehyde or keto group of donors 1.2.1 With NAD or NADP as acceptor 1.2.2 With cytochrome ~s an acceptor 1.2.3 Wi~h O~ as acceptor 1.2.4 With lipoate as acceptor 1.2.99 With other acceptors 1.3 Acting on the CH-CH yroup of donors 1.3.1 With NAD or NADP as acceptors 1.3.2 With a cytochrome as an acceptor 1.3.3 With 2 as acceptor 1.3.99 With other acceptors 1.4 Acting on the CH-NH2 group of donors 1.4.1 With ~D or NADP as acceptor 1.~.3 With 2 as acceptor 1.5 Acting on the G-NH group of donors ~ :
1.5.1 with NAD or NADP as acceptor 1.5.3 With 2 as acceptor 1.6 Acting on reduced NAD or NADP as donor 1.6.1 With NAD or NADP as acceptor 1.6.2 With a cytochrome as an acceptor 1.6.4 With a disulfide compound as acceptor 1.6.5 With a quinone or related compound as acceptox 1.6.6 With a nitrogeneou~ group as acceptor 1.6.99 With other acceptors 1.7 Acting on other ~itrogeneous compounds as donors 1.7.3 With O~ as acceptor 1.7.99 With other acceptors 1.8 Acting on sulfur groups of donors 1.8.1 With NAD or NADP as acceptor 1.8.3 With 2 as acceptor ~; ' ' "' ' '' :

' i ' ' 1.8.4 With a disulfide compound as acceptor 1.8.5 Wi~h a guinone or related compound as acceptor 1.8.6 Wi~h nitrogeneous group as acceptor l.9 Acting on heme groups of donors 1.9.3 With 2 as acceptor 1.9.6 With a nitrogeneous group as acceptor 1.10 Actiny on diphenols and related s~stances as donors 1.10.3 With 2 as ac~eptors l.ll Acting on H2O2 as acceptor 1.12 Acting on hydrogen as donor 1.13 Acting on single donors with incorporation of oxygen (oxygenases) 1.14 Acting on paired donors with incorporation of oxygen into one donor (hydroxylases~
1.14.1 Using reduced NAD or NADP as one donor 1O14.~ Using ascorbate as one donor 1.14.3 Using reduced pteridine as one donor 2. Transferases 2.1 Transferring one-carbon group~s 2.1.1 Methyltransferases 2.1.2 -Hydroxymethyl-, ormyl- and related transferases 2.1.3 Carboxyl- and carbamoyltransferases 2~ Amidinotransferases 2.2 Transferring aldehydic or ketonic residues 2.3 Acyltransferases 2.3.1 Acyltran6ferases 2.3.2 Aminoacyltransferases 2.4 Glycosyltransferases 2.4.1 Hexosyltransf~rases 2.4.2 Pentosyltransferases 2.5 Transfexring alkyl or related groups 2.6 Transferring nitrogenous groups 2.6.1 Aminotransferases 2.6.3 Oximinotransferases .~ .

3;~
5~L
2 . 7 Transferring phosphorus~containing groups 2.7.1 Phosphotransferases with an alcohol group as acceptor 2.7.2 Phosphotransferases with a carboxyl group as acceptor 2.7.3 ~hosphotransferases with a nitrogeneous group as acceptor 2.7.4 Phosphotrans*erases with a phospho-group as acceptor 2.7.5 Phosphotransferases, apparently intromolecular 2.7.6 Pyrophosphotranserases 2.7.7 . Nucleotidyltr`ansferases 2.7.8 Transferases for.other sl~stituted phospho-groups 2.8 Transferring sulfur-containing groups 2.8.1 Sulfurtransferases 2.8.2 Sulfotransferases 2.8.3 CoA-transferases 3. Hydrolases 3.1 Acting on ester bonds 3.1.1 Carboxylic ester hydrolases 3.1.2 Thiolester hydrolases 3.1.3 Phosphoric monoester hydrolases ~ :
3.1.4 Phosphoric diester hydrolases -3.1.5 Triphosphoric monoester hydrolases 3.1.6 Sulfuric ester hydrolases 3.2 Acting on glycosyl compounds 3.2.1 Glycoside hydrolases 3.2.2 ~ydrolyzing N-glycosyl compounds 3.2.3 Hydrolizing S-glycosyl compounds 3.3 Acting on ether bonds 3.3.1 Thioether hydrolases 3.4 Acting on peptide bonds (peptide hydrolases) 3.4.1 a~Aminoacyl-peptide hydrola~es 3.4.2 Peptidyl-aminoacid hydrolases 3.4.3 Dipeptide hydrolases 3.4.4 Peptidyl-peptide hydrolases ,1.
. ' :

.

3.5 Acting on C-N bonds other than peptide bonds 3~5.1 In linear amides 3.5.2 In cyclic amides 3.5.3 In linear amidines S 3.5.4 In cyclic amidines 3.5.5 In cyanides 3.5.99 In other compounds 3.6 Acting on acid-anhydride bonds 3.6.1 In phosphoryl~containinc3 anhydrides 3.7 Acting on C-C bonds 3.7.1 In ketonic substances 3.3 Acting on halide bonds 3.8.1 In-C~halide compounds 3.8.2 In P-halide compounds 3.9 Acting on P-N bonds :
4. Lyases 4.1 Carbon-carbon lyases 4.1.1 Carboxy-lyases 4.1.2 Aldehyde~lyases 4.1.3 Ketoacid-ly~ses 4.2 Carbon-oxygen lyases 4.2.1 Hydro-lyases 4.2.99 Other carbon-oxygen lyases 4.3 Carbon-nitrogen lyases 4.3.1 ~mmonia-lyases 4.3.2 Amidine lyases 4.4 Carbon-sulfur lyases 4.5 Carbon-halide lyases 4.99 Other lyases 5. Isomerases 5.1 Racemases and epimerases 5.1.1 Acting on amino acids and derivatives 5.1.2 Acting on hydroxy acids and derivatives 5.1.3 Acting on carbohydrates and derivatives 5.1.99 Acting on other compounds 5.2 Cis-trans isomeras~s , ~l3~3~
5~
5.3 Intramolecular oxidoreductases 5.3.1 Interconverting aldoses and ketoses 5.3.2 Interconverting keto ancl enol groups 5.3.3 Transposing C-C bonds 5.4 Intramolecular transferases 5.4.1 Transferring acyl groups 5.4.2 Transferring phosphoryl groups 5.4.99 Transferring other groups 5.5 Intramolecular lyases 5.99 Other isomerases 6. Ligases or Synthetases 6.1 Forming C~O bonds 6.1.1 Aminoacid~RNA ligases 6.2 Forming C-S bonds 6.2~1 Acid-thiol ligases 6.3 Forming C-N bonds 6.3.1 Acid-ammonia ligases (c~mide synthetases) 6.3.2 Acid-aminoacid ligases (peptide synthetases) 6.3.3 Cylo~ligases 6.3.4 Other C-N lig~ses 6.3.5 C-N ligases with glutamine as N-do~or 6.4 Forming C-C bonds Of particular interést will be enzymes which are in Class 1. Oxidoreductases and Class 3 hyrdolases, although enzymes of Class 2, Transferases, Class 4 Lyases and Class 5, Isomerases, can also be of interest in particular situatio~s.
The foll~wing table has specific subclasses of enzymes and specific enzymes within the subclass which are of particular interest. Among the oxidoreductases, -those in-volving NAD or NADP, oxygen or hydrogen peroxide are of particular interest. Among the hydrolases, those in~olving phosphate and glycosides are of particular interest.

.

`3;~

TABLE III
1. Oxidoreductases 1.1 Acting on the CH-OH group of donors 1.1.1 With NAD or NADP as acceptox 1. alcohol dehydrogenase 6. glycerol dehydrogen~se 27. lactate dehydrogenase 37. malate dehydrogenase 49. glucose-6-phosphate dehydrogenase 1.1.3 With 2 as acceptor 4. glucose oxidase galactose oxidase 1.2 Acting on the aldehyde or keto group o~ donors 1.2.1 With NAD or NA~P as acceptor lS 12. . glyceraldehyde-3-phosphate dehydrogenase .
1.2.3 With 2 as acceptor 2. xanthine oxidase luciferase 1.4 Acting on the CH-NH2 group of dcnors 1.4.3 With 2 as acceptor 2. L-amino acid oxidase 3. D-amino acid oxidase 1.6 Acting on .reduced NAD or ~ADP as donor 1.6.99 With other acceptors diaphorase 1.7 Acting on other nitrogeneous compou.nds as donors 1.7.3 With 2 as acceptor 3. Uricase 1.11 Acting on H2O2 as acceptor 1.11.1 6. catalase

7. peroxidase 2. Transferases 2.7 Transferring phosphorus--containing groups 2.7~1 Phosphotransferases with CH~OH
as acceptor l. hexokin~se `3~

2. glucokinase 15. ribokinase 28. triokinase 40. pyruvate kinase 52.7.5 1. phosphoglucomutase 3. Hydrolases 3.1 Acting on ester bonds 3.1.1 Carboxyllc ester hydrolases 7. cholinesterase

8. psuedo cholinesterase 3.1.3 Phosphoric monoester hydrolases 1. alkaline phosphatase 2. acid'phosphat:ase

9. glucose~'6-phosphatase 11. fructose diphosphatase 3.1.4 Phosphoric diester hydrolases l. phosphodiesterase 3. phospholipase C
3.2 Acting on glycosyl compounds 203O2.1 Glycoside hydrolases 1. alpha amylase 2. beta amylase 4. cellulase 17. muramidase 18. neuraminidase 21. beta g:Lucosidase 23. beta galactosidase 31. beta glucuronidase 3~. hyaluronidase 303.~.2 ~ydroly2ing N-glycosyl compounds 5. DPNase 4. Lyases 4.1 Carbon carbon lyases 4.1.2 Aldehyde lyases 13. aldolyase 4.2.1 ~ydro-lyases 1. carbonic anhydrase ~3~

5. Isomerase 5.4 Intramolecular transferases 5.4.2 Transferring phosphoryl group trio~e phosphate isomerase Nonen~ymatic catalysts may also find use, but will normally no-t be employed by themselves, but in conjunction with an enzymatic catalyst. Therefore, their use will be discussed in conjunction with the preferential production of the signal generating compound at the solid surface.
Of particular interest in the subject invention is the use of coupled catalysts, usually two or more enzymes, where the product of one enzyme serves as the substrate of the oth~r enzyme. One or more en2ymes are bound to the surface, while one enzyme is always bou~d to a mip. Alterna-tively, two enzymes can be hound to a mip, with or without an additional enzyme bound to the surface. The solute will be the substrate of any one of the enzymes, but preferably of an enzyme bound to the surface. The en2ymatic reaction may involve modifying the solute to a product which is the sub-strate of another enzyme or production of a compound whichdoes not include a substantial portion of the ~olute, which serves as an enz~me substrate. The first situation may be illustrated by glucose-6-phosphate being catalytically hydrolyæed by alkaline phosphatase to glucose, wherein glucose is a substrate for glucose oxidase. The second situation m~y be illustrated by glucose being oxidized hy glucose oxidase to provide hydrogen peroxide which would enzymatically react with the si~nal generator precursor to produce the signal generator.
Coupled catalysts can al60 involve an enzyme with a non-enzymatic catalyst. The enz~me can produce a reactant which undergoes a reaction catalyzed by the non~enz~matic catalyst or the non~enzymatic catalyst may produce a sub-strate (includes coen~ymes) for the enzyme. For example, Meldola blue could catalyze the conversion of NAD and hydro-quinones to NADH which reacts with FMN o~idoreductase a~d bacterial luciferase in the presence of long chain aldehydes to produce light.

.

A wide variety of nonenæymatic catalysts which may be employed in this invention are found in U.S. patent ~,160,645l issued July 10, 1979. The nonenzymatic catalysts employ as reactants a first compound which reacts by a 1-electron transfer and a second compound which reacts by a 2-electron transfer, where the two reactants are capable of reacting with each other slowly, if at all, in the absence of the catalyst.
Various combinations of enzymes may be employed -to provide a signal generating compound at the surface. Par-ticularly, combinations of hydrolases may be employed to produce an insoluble signal generator. A single exohydrol-ase may act in a substantially equivalent manner to an enzyme pair by employing the appropriate substrate. Alter-natively, combinations oE hydrolases and oxidoreductases canprovide the signal generator. Also, combinations of oxidore-ductases may be used to produce an insoluble signal generator.
The following table is illustrative of various combinations which may be employed to provide for preferential production of the signal generating compound at the surface. Usually there will be a preferred catalyst at the surface since, as indicated previously, by appropriate ~hoice of the catalyst at the surface, a greater number or reagents may be combined in a sing]e formation In the following table the first enzyme is intended to be bound to the surface and the second enzyme to a mip, although in particular situations it may be desirable to reverse their positions.

~ , - -,~

~ .

INTERRELATED TWO ENZYME SYSTEMS

First Second Signal Enzyme ~ Solute GeneratiQn -~. Galactose horse radish ~-D-galactose 4-Cl-1-5oxidase peroxidase naphthol dye 2. uricase horse radish urate o-dianisidine peroxidase dye 3. glucose microper- ~-D glucose bis-toluidine oxidase oxidase . dye 4. esterase ~-glucur- 2,2-bis(3'- 3',3"-clichloro-onidase chloro- phenolphthalein 4'-glucurony-loxyphenyl) phthalide choline chloride ester 5. alkaline peroxidase 4-Cl-1- 4-Cl-l-phosphatase naphthyl naphthol dye phosphate 20 6. hexokinase glucose-6- glucose iodonitro-phosphate triphenyl dehydrogenase formazan 7. alkaline ~-galactosi- o7 - ( ~-D-gal- 4-alkylum-phosphatase dase actosidyl-6'- belliferone phosphate) 4-alkylumbel-liferone ~3~

~2 INTERRELATED TWO ENZYME SYSTEMS-continued First Enzyme __ Reactlons 1. Galactose 1. galactose ~ 2 ~ D-galactono-~-lactone 5oxidase 2 2 2. H2O2 ~ ~~Cl-l-naphthol ~ dye 2. uricase 1. urate + 2 ~ allantoin ~ H202 2. ~22 ~ o-dianisidine -~ dye 3. glucose 1. glucose ~ 2 ~ D-glucono-~-lactone 10oxidase . ~2Q2 2. H2O2 +`bis-toluidine ~ dye .
4. esterase 1. 2,2~bis~3'-chloro-4'-glucuronyloxyphenyl) phthalide choline chloride ~ 2,2-bis (3'-chloro-4'-~lucuronyloxyphenyl)-phthalide 2~ 2,2-bis(3'-chloro-4'-glucuxonyloxy-phenyl3phthalide ~ 3',3"~dichloro-phenolphthalein 5. alkaline 1. 4-Cl-l-naphthyl phosphate ~ 4 Cl-l-20phosphatase naphthol 2. 4-Cl-l-naphthol ~ dye 6. hexokinase 1.. glucose ~ ATP ~ glucose-6-phosphate 2. glucose-6-phosphate + NADP ~ NADPH
phenazine methosulfate ~ N~DPH -~
triphenyltetrazolium chloride -~ formazan 7. alkaline 1. o7-~ D-galactosidyl-6'-phosphate)-phosphatase 4-alkylumbelliferone ~ o7~ D-galactosidyl) 4-alkylumbelliferoIle 2 . o7 - ~ ~-D-galactosidyl) 4-alkylumbel~
liferone ~ 4-alkyl~mbelliferone ., : " -,' , ~3~3~

INTERRELATE:D ENZYME AND NON-ENZYMATI C CATALYST SYSTEMS

Enzyme on Signal mip Catal~st Solute eneration 1. G-6-PDH Meldola N.AD formazan blue 2. lactate phenazine NAD benzyl-violo-dehydrogen- methosulfate gen dye ase 3. 3-hydroxy pyocyanine NAD forma2an butyrate dehydro-genase Precursor to signal generator may be covalently bonded to solid surface.

INTERRELATED ENZYME AND NON-ENZYMATIC CATALYST SYSTEMS
continued Enz~me on ~ æ___ Reactions 1. G-6~PDH 1. G-6-P + NAD ~ glucuronate-6-P + NADH
2. NADH ~ triphenyltetrazolium ~ NAD
formaxan 2. lactate l. lactate + NAD ~ pyruvate + NADH
dehydrogen- 2. NADH -~ ben~yl violoyen ~ NAD + dye a~e 25 3. 3-hydroxy 1. 3-hydrox~butyrate + NAD ~ acetoacetate butyrate + NADH
dehydro- 2. NAD~ + triphenyltetrazolium ~ NAD +
genase 0rma2an Quite obviously, many of the dyes indicated above may be substituted with other dyes which have the proper solubility requirements or which can be modified to have the proper solubility requirements for the subject invention. In addition, it should be appreciated, that by having a high localized concentration of the dye, the dye will rapidly bind to the surface. In addition, any incremental amount of dye which diffuses from the bulk solution to the surface will not significantly affect the amount of dye which precipitates on the surface. Depending upon the nature of the dye, ei~her light absorption by the dye or, if fluorescent, light emis-sion may be measured. Instead of dyes, electroactive com-pounds may be produced and electrical properties at the surface measured.
Instead of a chemical react.ion of an enzyme product to produce the signal generating compound, the environment of the enzyme product can be selectively modified, upon binding to the surface, 50 as to produce the signal generating com-pound. For example, one could hydrolyze an ester or ether to produce an insoluble colorless form of a p~ sensitive dye at the surface. The local pH at the surface will be made sub-stantially different from the bulk solution by having changed groups on the surface. By employing a signal genexating compo~md which is sensitive to proton concentration, the observed signal from the product bound to the surface would differ greatly from the product in the bulk solution or liguid phase. Fluorescer quencher pairs may also be employed where the solute produces an insoluble quencher molecule, which binds to the surface. Increasing amounts of the quencher molecule on the surface will result in substantially decreased fluorescence from the fluorescent mQlecules bonded to the surface.
Besides acid-base effects and fluorescex and quencher pairs, other interactions may include enzyme i~hibitor~enzyme combinations, medium effects caused by hydrophobic binding, redox reactions, covalent coupling to form surface bound dyes, or the like.

:, :

'' " ' ~

.

`3~

One can further enhance the differentiation between the concentration of -the signal generator at the solid sur-face as compared to the signal generator in the solution, by having a scavenger for a member of the signal producing system. The role ~f the scavenger is to int:eract with a component of the signal producing system to inhibit the functioning of the component in the production of the detect-ible signal. The scavenger employed can act: in a variety of ways.
The first way is -to employ a scavenger for the signal generator which interacts with the signal generator to inhibit its formation of a signal. This inhibition can be as a result of a chemical reaction or specific or nonspecific binding. So far as a chemical reaction, a wide variety of chemical reactions can be employed depending upon the nature of the signal generator. For example, if the dye goes from a leuco to a colored form by virtue of an oxidation-reduction reaction, by providing fox reversing the reaction in the bulk solution, the colored ~orm can be substanti.ally minimized in the bulk solution. The chemical scavenger employed should either react with the signal generator on the surface ex-tremely slowly or not at all. Convenient scavenger~ would be enzymes which would reverse the redox reaction or antibodies which would modify the absorption or emission characteristics of a dye. Enzymes or antibodies could be employed which are monomeric or pol~meric e.g. bound to particles, so as to reduce their ability to interact with the si~nal generator on the surface.
Instead of scavengers for the signal generator, one may employ a scavenger for a different member or intermediate of the signal generating system. One can utilize the steric bulk of the surface to discriminate between càtalyst bound to the surface and catalyst in -the bulk solution. By employing inhibitory antienzyme bound to particles, en~ymes in the bulk solution would be inhibited from reacting when the antien~yme bound to the enzyme, while the enzyme bound to the surface would be free to react. Similarly, one could provide other inhibitors bound to particles wi-th which the enzyme reacts resulting in destruction o the enzyme activity.

~3~3~

Where two enæymes are involved so that an inter-mediate product is involved, one could employ as the scaveng-er a reactant which destroys the intermediate. For example, where an .intermediate product is hydrogen pleroxide, by adding catalase, the hydrogen peroxide would be destroyed in the bulk solution. At the surace, however, thlere would be more efective competition by the relatively concentrated catalyst bound to the surface with ~he catalase for the hydrogen peroxide. This competition could be made even more favorable by attaching the catalase to particles that are sterically excluded from the surface. With different systems, different technigues can be employed, using the versatility of the subject system to achieve the desired effect.
Finally, one can provide:for-a compound bonded to the surface which will interact with a product o the cata-lyzed reaction. For example, one can produce a compound which will react with the compound on the surface to change the compound rom the leuco form to the colored form. Illus-trative of such a technique would be to oxidize NAD~ with a nonenzymatic catalyst and a tetrazolium salt bonded to the surface. The catalyst could be, for example, phenazine methosulfate or Meldola Blue. The NAD~ would react with the catalyst which would promptly react with the tetrazolium salt on the surface to form the dye. One could conveniently couple this with a scavenger, such as an oxidant which reoxidized the reduced catalyst, so that any reduced catalyst which was formed in the bulk solution would be rapidly destroyed. A significant alternative would be, for example, 1,7-naphthalenediol bound to the surface which captures aminoantipyrene oxidation product produced by HRP. Less chromogenic nucleophiles could act as scavengers.
The next element of the signal producing system is the solute. The solute will be the initial reactant subject to catalytic transformation to a product. As already diso cussed, the product can play a number of different roles.
The product may be the signal generator which becomes bound to the solid surace. Alternatively, the product may be an intermediate which serves as a substrate for a second .

~31~3%

catalyst, which further transforms the product to provide the signal generator. Alternatively, the solute can undergo a reaction which leads to a compound which then react~ with ~nother compound to produce the signal geneI-a-tor. The other compound may be free in solution or bound to the solid sur~
face, with the reaction being either catalyzed or uncatalyzed.
Ancillary Materials Various ancillary materials will i-re~uently be employed in the subject assays. Particularly, en~yme sub-strates, cofactors, ~ctivators, scavengers, inhibitors or the like may be included in the assay medium.
In addition, buffers will narmally be present, as well as stabilizers. Frequently in addition to these addi-tives, additional proteins may be included, such as albumins;or surfactants, particularly non-ionic surfactants, e.g.
polyalkylene glycols, or the like.
Compositions Novel compositions and solid test films or strips are provided, as well as combinations of reagents for use in the determination of a wide variety of analytes. Of particu-lar interest are haptens having physiologic activity, coval-ently bonded to a solid porous support to which is also covalently ~onded an enzyme, particularly a redox enzyme or a hydrolase. The solid supports are used in conjunction with a receptor to which is bonded an enzyme, where the enzyme-bound-receptor employs the product of the enzyme-bound~solid support as a substrate. Included with the enzyme-bound-receptor is the suhstrate for the enzyme-bound solid support.
Impregnated in the solid support may be buffers, s~strates and cofactors for the enzyme on the solid supp~ort othex than the substrate or cofactor combined with the enzyme-bound-receptor. The amQUnts of the various reagents are optimized to enhance the sensitivity of the assay for the analyte.
For an antigenic analyte, either the receptor ~or the antigen or the antigen may be covalently bonded to the solid support along wi~h an enzyme. The solid support con-taining the receptor or antigen and the enzyme is analogous to the solid support with ~he hapten.

Alternatively, a solid support can be employed haviny a mono- or polyepitopic antigen or receptor bound to it in conjunction with a covalently bonded electrophilic or nucleophilic coupler. The coupler can react wi~h its appro-priate partner. For example, with a nucleophilic couplerbound to the support, oxidized forms of developers, such as aromatic amines may be employed as coupling partners to form dyes. The reduced form of the dye may be used as the solute in conjunction with an enzyme-bound-mip, where the enzyme is lQ an oxidoreductase, such as peroxidase. The peroxidase will oxidize the reduced form of the developer e.g. aromatic amine, which will react with the coupling agent on the sup-port to produce a dye. Illustrative coupling agents include phenols, ~-diketones, pyrazalones, and the like.
Finally, a solid support can he employed having a hapten, antigen or antibody in conjunction with a compound covalently bonde~ to the solid support which can react with -the reduced form of Medola Blue, phenazine methosulfate or methylene blue to go from a leuco form to a colored form.
The strips will be used in conjunction with the oxidized form of the aforementioned reductants, an enzyme-antibody conju~
gate, where the enzyme desirably produces NADH or NADP~ or other reductant which will react with the aforementioned catalytic reductants, which in turn will react with the lèuco 5 form of the dye on the solid support.
EXPERIMENTAL
The following examples are offered by way of illus-tration and not by way of limitation.
All percents and parts not o~herwise indicated are by weight, except for mixtures of liquids which are by volume. When a solvent is not indicated, water is intended.
All temperatures no-t otherwise indica-ted are centigrade. The following abbreviations are employed:
C~M-03-carboxymethyl morphine; HRP-horse radish peroxidase; NHS-N-hydroxy succinimide; EDCI-N-ethyl N'-(3-dimethylaminopropyl) carbodiimide; DMF-N,N-dimethyl formamide; THF~tetrahydrofuran; BSA-bovine ~erum albumin;
HIgG-human immunoglobulin G; THC-tetrahydrocannabinol derivative; RT-room temperature; G0-glucose o~ida~e.

3~

Ex. 1. Morphine Horseradish Peroxidase (~RP) ~onju~ate _ __ Into a reaction flask was combined lO~moles O -carboxymethylmorphine, ll~moles of N-hydroxy succinimide and 12~moles of EDCI in a to~al volume of about l~.lml in DMF. After combining ~he reagents, the mixture was flushed with nitrogen and stirred overnight in a co:Ld room. To 0.5ml HR~ ~2mg) in 50mM agueous sodium carbonate l~p~9.5) was added 150ml DMF, followed by 300~1 of the above ester solution and the mixture allowed to stand overnight at 4. The reaction mixture was then applied to a 2x30cm column of G50 Sephaclex and eluted with 0.05M tris, pH7O6, 0.lM KCl and the protein monitored. The fractions in the void volume were pooled to provide 0.5ml having a concentration of 0.2mg/ml. By employ-ing a radioactive tracer, the morphine/HRP molar ratio wasfound to be 1.86 with a concentration of HRP of 200~g/ml.
Ex. 2. Protein Couplin~ to Paper Filter Disk~Morphine The following is -the exemplary protocol employed for protein coupling to a paper support. Whatman #2 filter paper disks 7cm dia. were activated in O.lM sodium periodate for 5hrs. at room temperature. After washi~g with water extensively, and drying in THF, lml of the apprvpriate protein solution in 0.2M borate, pH8.5, 0.5M NaCl, O.lM
NaBH3CN was added to the disks and the mixture allowed to stand overnight at 4. To the mixture was -then added 1.4ml 50mM Bicine buffer, pH8.5, containing 2mg NaBH4 and the mixture allowed to stand for 3hrs. at room temperature, followed by termination by washing the disks in lM borate, pH8.5, 50mM Bicine, 0.2M KCl. The wash was about 20ml and the protein in the wash was determined, with the amount of protein bound to the disk determined by the difference.
The following table indicates the different protein solutions employed and the concentr~tion of protein on various d1sks in ~g/cm2.

3~

TABLE IV

Total Protein Protein in Coupled to disc 1.5ml of Solution1 ~g/cm2 lOmg Ab~ 24.3 lOmg Ab~, 5A28oG0 45.6 5mg AbM~ 5A~8oG0 6.53 Y ~IgG' 5A280~ 7.39 g ~(RIg)' 280 8.69 0 ~ AbM ~ antibody to morphine 5A28oGO - glucose oxidase at a concentration having an absorption of 5 at A280nm per cm AbHIgG ~ antibody to human ~-globulin G
AbG(RIg) ~ antibody to rabbit r-globulin G from goat antisera In order to d~monstrate the subject invention, a number of determinations were carried out to determine the ~.
effect of having ligand analyte present in ~he assay medium.
The following sample solutions were prepared. The disk is indicated by the particular protein(~) bound to the paper.

TABLE V

HRP-M2 CMM3RIg-HRP4 RIg5 # aperl ,ul ~ ~ ,ul 2$ 1. AbM 30 0 -2. AbM 30 0 3. Ab~ 30 30 4. AbM 30 30 - -5. AbMGO(2:1) 30 0 - -6. AbMGO(2:1) 30 0 - -7. ~bMG0~2:13 30 30 - -8. Ab~GO(2:13 30 30 , .

`3;~

TABLE V
~ continued ) HRP-M2C~3 RIg~~RP4 RIg5 Paper~
9 AbGARGO - ~ 4~

10 AbGARG0 ~ ~ 44

11 Ab~ARG0 4 10

12- AbG~R&O ~ ~ 44 10 1 Whatman filter paper (~6mm dia) AbM ~ morphine antisera (-~6.08 ~gAb per disk) AbMGO(2:1) - morphine antlse`ra plus glucose oxidase 2:1 mole ratio in reaction medium (7.6,ugAb per disc) AbGARGO - goat anti(rabbit IgG) plus glucose oxidase 1:1 mole ratio in xeaction medium (~2.2~gAb per disk) 2 ~RP~M - morphine conjugated to horse radish peroxidase and product diluted (20yg/ml~
3 CMM - 03-carboxymethyl morphine (90~g/ml) 2 4 Rig-HRP - rabbit immunoglobulin G conjugated to horse radish peroxidase (13.8~gAb/ml) RAb~IgG - rabbit immunoglobulin ~ ~

The protocol was as follows. The disk and CMM or RIg were combined employing 0.94~1 of buffer, the buffer being 50mM tris, pH7.6, lOOmM KCl, and 0.lmg/ml BSA. The mixture was incubated for 5hrs followed by the addition of HRP-M or RIg-H*P in lml of the appropriate reaction buffer to ~he incubation buffer or the disk was removed from the incu-bation buffer, washed wi~h lml of water, and then combined with the HRP-M or RIg~HRP in the reaction buffer. Depending upon whether glucose oxidase was pxesent on the paper, the reaction buffers differed in that in the absence vf glucose oxidase, 20~1 of 90mM hydrogen peroxide was added in addition to sufficient o-dianisidine to provide 0.lmg/ml. In the presence of glucose oxidase, the buffer was made 50mM in glucose and no hydrogPn peroxide was added.

. ~ ':

... : :
: : : . . . :.

~.~.3~

The odd number examples were carried out retaining the disk in the incubation buffer and adding reaction buffer, while the even number~d examples were carried out with -~he removal of the disk from the incubation buffer, and washing 5 and then combining the disk and reaction buffer.
In each case, lml of the reaction buffer was employed, and in the odd numbered examples, a 60min incuba-tion was employed. In the Exs. 1, 3, 5, and 7, 20~1 of 3.9mg/ml catalase (30,000U/mg3 was also included, while in Exs. 9 and 11 only 10~1 of the catalase solution was included. In the even numbered examples, no catalase was added.
In each of Exs. 1, 5, and 9, the disks were darker than the comparable Exs. 3,-7, and 11 respectively, showing that the presence of a ligand did allow for dis~rimination in result.
In the even numbered assays, the reactions for 2, 4, 6 and 8 were carried out for 5mins, while for 10 and 12, lOmins. The results were far more dramatic with the even numbered examples, where the disk was clearly white in the presence of the ligand and a dark brown in the absence of the ligand.
The results clearly demonstrate that one can assay for a ligand, both haptenic and antigenic, or a receptor,~by employing a catalyst bound to a mip, which becomes distrib-uted between a surface and the bulk assay medium in propor-tion to the amount of ligand or receptor present in the medium. In the subject situation, an insoluble signal gener~
ator is produced which becomes bound to the surface and allows for measurement of the signal in relation to the amount of analyte in the assay medium.
In the next study, the effect on the amount of signal genera-tor produced in relation to varying concentra-tions of analyte was evaluated. In effect, a standard curve was prepared, relating the signal generator produced on the surface to the amount of analyte in the medium. The protocol was as follows.

3~

To tubes containing 500~1 bufer (50mM tris, pH7.6, 200mM KCl, 2mg/ml BSA), was added 20~l1 of the ~ -morphine conjugate (0.2~g) followed by 20~1 of a O -carboxymethylmorphine solution at varying concentrations.
To the tube ~as then added a 6mm disk of ~b~GO (2.1) and the mixture incubated a-t 3hrs at room temperature.
The disk was then developed in three different ways. In the first way, the supernatant was removed from the disk, lml buffer added and removed. Then, lml of the devel~
opment buffer (lOOmM phosphate, p~6.0, 200mM KCl, O.lmg/ml o-dianisidine~ and 10~1 90mM hydrogen peroxide were added and the mixture allowed to react for 5mins, followed by washing;
in the second method, the same procedur2 was employed, except that 2ml of buffer was added which did not contain the hydro gen peroxide, but was 15mM in ~~D glucose'~ The reaction was allowed to proceed ~or 30mins., followed by removing the disk, washing and drying. In the third technique, the second technique was repeated, except that 10~1 of a 3.9mg/ml catalase solution was added.
In each case, there was a ste~dy progression of increased darkness of the solid surface in going from the stock solution of 90~g/ml of 03-carboxymethylmorphine through 1:5 serial dilutions to a final amount of 1.6xlO 12 mole.
The midpoint was found to be 1.2xlO 10 mole or about 40ng of ~5 0 -carboxymethylmorphine.
A sesond series of studies were performed using morphine as an exemplary ligand. I'his study involved dif-ferent sample fluids which would provide varying backgrounds when performing the assay. The reagents were prepared as ollows.
Ex. 3. Protein Coupllng to Paper Filter Disk-Morphine In O.lM NaI04 was incubated l~.Scm of Whatman #2 paper for Shrs at RT. The paper was then incubated in lM
ethylene glycol for 20min followed by washing with 6Q of deionized water. To the paper was then added a total of 1. 8ml of the appropriate protein solutions in 50mM borate, 3~

0.2M NaCl, pH8.6 and the paper contacted with the protein solution for 2hrs at RT~ The following gives the composi~
tions of th~ protein solutions.
PROTEIN SOLUTION', AbM G o 1 BSA
_m~ protein (1) 4 (2) 1 1 3 (3) 0.~5 1 3.75 (4) 1 4 (5) 1 0.25 3.75 Absorbance per cm at 280nm .
To the paper was then added 2ml 2mg/ml NaBH4 in the same buffer and the mixture allowed to stand for lhr at E~T.
The paper was then washed with water and buffer, then im-mersed in a solution of 5mg/ml BSA, 15% sucrose and removed and lyophilized.
For US2 in assays, 3/8" disks were punched and the disks incubated in urine for 7min at RT, where the urine h~d either no morphin~ or 100ng/ml morphine. The disks were then transferred to lml of developer solution: 0.1mg/ml 4-Cl-l-naphthol, 2mg/ml BSA, 50mM glucose, 0.1M PO4, pH7.0, 0.2M
NaCl and 0.1mg/ml o-dianisidine. To the 601ution was then added 4~1 HRP-M (20~g/ml) and the mixture allowed to stand for 30min at RT and the tests repeated with 15~1 HRP-M and a reaction time of 60min.
In both cases ~he presence of the morphine was clearly detectable for (1) and (4), with less difference with the o~her samples. Thus, the assay is able to detect minute amounts of morphine in the complex proteinaceous urine mixture.
The next study was to determine whether morphine could be detected in milk. The above procedure was repeated employing lml raw whole milk with and without 100ng/ml morphine. The procedure was varied by employing 100~1 oE
2~g/ml ~P-M and 10~1 of 3.9mg/ml catalase and incubating for 60min at RT with a developer solution of the following com-position: ~OmM bicine, p~8.0, 200mM KCl, ~mg/ml BSA, 50mM
~-D-glucose and O.lmg/ml 4-Cl-l-naphthol. The diference between the samples with and without morphine was clearly detectable using disks prepared employing 1:1 of the AbM:GO
solution~ (see Table IV).
In -the nex~ study, -the use of a solid surface conjugated with a mip W2S employed for the determination of HIgG. In 0.5ml buffer containing 20~1 of an appropriate HIgG
concentration were incubated Smm paper disks (~bHIgGGO) for 3hrs at room temperature. The supernatant was removed, the disks washed, and O.Sml buffer added plus 50~1 o a 13.8~g/ml solution of AbHIg (DAKO). The mixture was then incubated for 3hrs at room temperature followed by the addition of lml of lOOmM phosphate, 200mM KCl, 50mM glucose and 0.lmg/ml o-dianisidine. Afker 30mins at room temperature, the disks were removed and washed, demonstrating a clear progression in the color of the disks in going from 0.16 -to 20~g of HIgG.
E~. 4. THC-HRP Conjugate Into a reaction flask was introduced O.l9ml of a 0.04M solution of the NHS ester of O~carboxymethyl oxime of 7,9,12-hexahydro-6,6-dimethyl-g-oxo-3-pentyldibenzo[b,d]
pyran~l-ol in DMF, 0.28ml ~RP (1.5mg), O.lml lM Na2C03, p~9.5, and 0.3ml H20 and the mixture stirred o~ernight.
After centrifuging to remove insoluble material, the supernatant was dialyzed against O.lM NaHCO3, 0.5M NaCl (4Q~.
The residue was chromatographed on a 20~1.5cm Sephadex G50 column in 50mM tris, pH7.6 J O.2ml and the void volume peak isolated, ~3ml, 0.26mg/ml.
~. 5. Protein Couplin~ to Paper Filter Dlsk-THC
The following protein solutions in 50mM borate, pH8.5, 0.2M KCl were employed: antitetrahydrocannabinol, IgG, ~3mg/ml; glucose oxidase, 18A2~0/ml.
Paper di~ks (Whatman ~1, 9cm~ were activated in O.lM NaI04 for 4hr at RT followed by washing with ~3Q H2O.
To an activated disk was added 2.5ml of a protein solution *Whatman is a trademark.

;

~: :

~3~

containing 3.83A280/cm glucose oxidase and 0.75mg of antisera for tetrahydrocannabinol and the mixture allowed to react for lhr at RT followed by the addition of 0~5ml of a 4mg/ml NaBH4 solution and the reaction mixture allowed to stand for 1.5hr at RT, turning the disk every 20min. The d:isk was then washed with 300ml 0.5~ NaCl, H2O (2x) and stored moist at 4.
In order to demonstrate the subject method for THC, disks (~6mm~ were employed with lml urine spiked with varying concentrations of THC. The urine sample was contacted with the disk for lOmin at RT and the disk then transferred to a developer solution containing 10~1 of a 1/100 dilution of the above THC-E~P in lml 50mM glucose, 50mM barbitol acetate buffer, pH7.6, O.lmg/ml 4-Cl-1-naphthol and r`action allowed to proceed at RT for lhr. At the end o-~ that term the disk in the urine containing 4.7ng/ml THC could be clearly distin-guished from the nega-tive urine sample.
In accordance with the subject invention, a simple sensitive technique is provided for determining analytes at extremely low concentrations and providing for a relatively permanent record of the result. The subject technigue allows for assays which can be carried out without highly trained personnel. Depending upon the rate at which the members of the immunological pair bind, various incubation times may be required, while development of the signal can be performed over relatively short times. Furthermore, the samplès can be used neat or with only minor dilution, so as to enhance the rate of binding of the members of the immunological pair to the solid surface.
The subject method provides for qualitative and quantitative determination of haptens, antigens and recep-tors, where standards can be performed to rela:te signal to concentration. Visual observation of the solid surface may be sufficient for qualitative or semiquantitative determina-tions, while instrumentation can be employed to enhance the ~uantitative nature of the result. A substantial variety of techniques may be employed to insure differentiation between the signal generator produced at ~he surface and any signal generator produced in the bulk solution. Thus, the signal gene.rated at -the surface can be directly related to the amount of analyte in the medium.
The subject method differs from prior art methods in providing a method for deter~ining extremely low concen trations of analytes by a simple protocol with a minimum number of steps and reagent formulations. Furthermore, the subject method involves a competition for binding sites or a cooperation between binding sites of mips to have a catalyst bind to a surface to provide a signal generating compound associated with the surface. This is achieved without requiring separation between the catalyst bound to the sur-face and the catalyst in solution.
Since the use of the s~r~ace as a "dip stick" for measuring analytes can be directed to use by non-technical people--even for use in the home -it is important th~t the method have as few indi.vidual steps as possible, be relative-ly foolproof, particularly by allowing for a contxol which can be carried out under substantially identical conditions, and have as few measurements as possible. The subject inven-tion fulfills these goals to a substantial degree.
Although the foregoing invention has been describedin some detail by way of illustration and example for pur-poses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope o~ the appended claims.

1 , ~
:, :
:, ,

Claims (19)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:-

1. A method for detecting the presence of an analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand;
said method involving (1) the partitioning of a catalyst having at least one substrate and bound to a mip--catalyst-bound-mip--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) a change in concentration of a signal generating compound associated with said surface, said change in concentratation resulting in varying the amount of reaction product produced by said catalyst bound to said surface;
said method comprising:
(a) combining in an aqueous assay medium, 1) said sample;
2) mip-bound-surface;
3) catalyst-bound-mip; and 4) the remaining members of the signal producing system, which system includes at least one catalyst including said catalyst-bound-mip, and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation or destruction of a signal generating compound associated with said surface and capable of producing a detectible signal, with the proviso that said signal producing system is completed not later than about the time of addition of catalyst-bound-mip to said surface, when said signal producing system consists essen-tially of said catalyst-bound-mip and its substrates;
(b) waiting a sufficient time for at least a portion of said catalyst-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample;
(c) determining the amount of detectible signal at said surface as a function of the amount of analyte in said sample.

2. A method for detecting the presence of an analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand;
said method involving (1) the partitioning of a catalyst having at least one substrate and bound to a mip--catalyst-bound-mip--between a surface and a liquid phase of an enzyme bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) a change in intensity of a detectible signal from a surface resulting from the change in concentra-tion of a signal generating compound associated with said surface, said change in intensity being related to the amount of reaction product produced by said enzyme bound to said surface;
said method comrising:
(a) combining in an aqueous assay medium, 1) said sample;
2) mip-bound-surface, wherein substantially 79a all of said mip-bound-surface is uniformly contacted with said sample;
3) enzyme--bound-mip; and 4) the remaining members of the signal producing system, which system includes at least one enzyme including said enzyme-bound-mip, and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in a change in amount of a signal generating compound associated with said surface and capable of producing a detectible signal, with the proviso that the binding of said enzyme-bound-mip to said surface and the formation of signal generating compound occurs at least a portion of the time concurrently, when the signal producing system consists essentially of said enzyme-bound-mip and its substrates;

b) waiting a sufficient time for at least a portion of said enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample;
c) determining the intensity of said detectible signal at said surface as a function of the amount of analyte in said sample.

3. A method according to Claim 2, wherein said signal producing system includes a second enzyme, where the product of said second enzyme is the substrate of said enzyme-bound-mip.

4. A method according to any of Claims 2 to 3, where a scavenger is included in the signal producing system which reacts with a component of said signal producing system to inhibit operation of said signal producing system in said liquid phase.

5. A method for detecting an haptenic ligand analyte in a sample suspected of containing said analyte, where said analyte is a member of an immunological pair (mip) consisting of haptenic ligand and homologous antiligand;
said method involving (1) the partitioning of an enzyme catalyst having at least one substrate and bound -to a haptenic ligand enzyme-bound-haptenic ligand--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to an antiligand-bound-surface, where said partitioning is in relation to the amount of haptenic ligand analyte in said sample; and (2) a change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye produced as a product of said enzyme of said enzyme bound-haptenic ligand, said change in concentration being in relation to the amount of insoluble dye produced by said enzyme bound to said surface;
said method comprising:
(a) combining in an aqueous assay medium, 1) said sample;
2) antiligand-bound-surface;
3) enzyme-bound-haptenic ligand; and 4) the remaining members of the signal producing system, which system includes at least one enzyme including said enzyme-bound-hapten ligand and one or more enzyme substrates which include a leuco dye or source of leuco dye which is capable of undergoing an enzyme catalyzed reaction catalysed by said enzyme-bound-haptenic ligand to produce an insoluble dye which precipitates on said surface, with the proviso that at least a portion of said binding of said enzyme-bound-ligand to said surface and formation of said insoluble dye occurs concurrently, when the signal producing system consists essentially of said enzyme-bound-haptenic ligand and said substrates;

b) waiting a sufficient time for said enzyme-bound-haptenic ligand to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of said insoluble dye associated with said surface in relation to the amount of haptenic ligand in said sample and c) determining the amount of detectible signal at said surface from said insoluble dye as a function of the amount of haptenic ligand in said sample.

6. A method according to Claim 5, wherein said signal generating system includes a second enzyme bound to said surface, wherein said second enzyme reacts with a solute to produce a product which is a substrate for said enzyme of said enzyme-bound-haptenic 1igand.

7. A method for detecting the presence of a polyepitopic antigenic analyte in a sample suspected of containing said analyte, where said antigenic analyte is a member of an immunological pair (mip) consisting of ligand and homologous antiligand;
said method involving (1) the partitioning of an enzyme catalyst having at least one substrate and bound to a mip-enzyme-bound-mip--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) a change in concentration of a signal generating compound associated with said surface where said signal generating compound is an insoluble dye, said change in concentration being in proportion to the amount of insoluble dye produced by said enzyme bound to said surface;
said method comprising:
(a) combining in an aqueous assay medium;
1) said sample;
2) mip-bound-surface;
3) enzyme-bound-mip, wherein one of the mips of said mip-bound-surface and enzyme-bound-mip is antiligand; and 4) remaining members of said signal producing system, which system includes at least one enzyme including said enzyme-bound-mip and wherein the enzyme catalyzed reaction of said enzyme-bound-mip results in the production of an insoluble dye as the signal generating compound, which precipitates onto said surface, when said enzyme-bound-mip is bound to said surface through the intermediacy of ligand-antiligand binding, with the proviso that when said signal producing system consists essentially of said enzyme-bound-mip and substrates for said enzyme at least a portion of said binding of said enzyme-bound-mip to said surface and production of said insoluble dye occurs concurrently;

b) waiting a sufficient time for said enzyme-bound-mip to bind to said surface through the intermediacy of ligand-antiligand binding and for a change in the amount of signal generating compound associated with said surface in relation to the amount of analyte in said sample; and c) determining the amount of detectible signal at said surface as a function of the amount of analyte in said sample.

8. A method according to Claim 7, wherein said signal producing system includes a second enzyme bound to said surface, wherein said second enzyme reacts with a solute to produce a product which is a substrate of said enzyme of said enzyme-bound-mip.

9. A method for detecting the presence of a ligand analyte in a sample suspected of containing said ligand where said ligand is a member of an immunological pair (mip) consisting of ligand and homologous antiligand;
said method involving (1) the partitioning of an enzyme having at least one substrate and bound to a mip--enzyme-bound-mip--between a surface and a liquid phase, where said partitioning is through the intermediacy of ligand-antiligand binding to a mip-bound-surface in relation to the amount of analyte in said sample; and (2) a change in concentration of a signal generating compound associated with said surface, where said signal generating compound is an insoluble dye, said change in concentration being in relation to the amount of insoluble dye produced by said enzyme bound to said surface said method comprising:
(a) combining in an aqueous assay medium 1) said sample;
2) mip-bound-surface, wherein said mip-bound-surface is substantially uni-formly contacted with said surface;
(b) incubating for sufficient time for ligand analyte to bind to said mip-bound-surface;
(c) after sufficient time for said incubation, adding substantially concurrently enzyme-bound mip; and (d) remaining members of said signal producing system, which system includes at least said enzyme of said enzyme-bound-mip and a solute which is capable of undergoing a catalyzed reaction to produce a product which results in the formation of a signal generating compound, which is an insoluble dye capable of producing a detectible signal, (e) incubating for sufficient time for said insoluble dye to precipitate on said surface in proportion to the amount of analyte in said sample; and (f) determining the amount of said detectible signal on said surface as a function of the amount of analyte in said sample.

10. A method according to Claim 9, wherein said mip-bound-surface to which said ligand analyte has bound is separated from said sample prior to adding said signal producing system.

11. A method according to Claim 9, wherein said signal producing system includes a second enzyme bound to said surface, said solute being the substrate of said second enzyme which results in the formation of a product which is the substrate of said enzyme of said enzyme-bound-mip.

12. A method according to Claim 10, wherein said signal producing system includes a scavenger in the liquid medium which reacts with a component of said signal producing system to inhibit operation of said signal producing system in said liquid phase.

13. A kit for use in an immunoassay comprising in combination a member of an immunological pair and a catal-yst bound to an insoluble surface, an enzyme bound to a member of an immunological pair, and a leuco dye which under-goes an enzymatically catalyzed reaction to produce an in-soluble dye capable of binding to said surface.

14. An article of manufacture useful in an immuno-assay and comprising a base having an insoluble surface and having an enzymatic or non-enzymatic catalyst covalently bonded thereto and a member of an immunological pair.

15. An article as claimed in claim 14, in which the base is bibulous.

16. An article according to claim 15, wherein said catalyst is an enzyme.

17. An article according to claim 16, wherein said enzyme is an oxidoreductase.

18. An article according to claim 17, wherein said enzyme is glucose oxidase.

19. An article according to claim 16, wherein said enzyme is a hydrolase.