CA1231048A - Inverted latency specific binding assay - Google Patents

Abstract

ABSTRACT

A specific binding assay composition and method for determining a ligand in a sample are disclosed. The composi-tion comprises (a) a binding partner for the ligand; (b) a detection system which has at least two components; (c) a selectively accessible vesicle having a surface-incorporated ligand or ligand analog and a first component of the detection system therein; (d) a substance which modifies vesicle accessibility in response to binding of surface-associated ligand or ligand analog and binding partner; and (e) at least one additional component of the detection system which is reactive with the first component to produce a detectable response which is reduced by association of the binding partner and vesicle modifying substance with the variabily accessible vesicle. A decrease in signal is measured upon reaction as compared to the signal of unreacted vesicles.

Description

I

INVERTED LATENCY SPECIFIC ENDUING ASSAY

BACKGROUND OF THE INVENTION
_ . .

Field of the Invention This invention relates to the field of specific binding assays, particularly immunoassay for determining substances of clinical interest. The development of specific binding assay techniques has provided extremely useful analytical methods for determining various organic substances of diagnose tic, medical, environmental and industrial importance which appear in liquid mediums at very low concentrations. Specific binding assays are based on the specific interaction between a ligand, i.e., a bindable analyze under determination, and a binding partner therefore i.e., receptor. The presence of the receptor can be used to effect a mechanical separation ox bound and unbound labeled analyze or can affect the label in such a way as to modulate the detectable signal. The former situation is normally referred to as heterogeneous and the latter as homogeneous, in that the latter technique avoids a separation step. Where one of the ligand and its binding partner is a h~pten or antigen and the other is a corresponding antibody, the assay is known as an immunoassay. See, generally, O'Dell and Dodd (Ens.), Principles_ of competitive Protein-in n assays, J. B. Lippincott Co., Philadelphia (1~71).

In conventional label conjugate specific binding assay techniques, a sample of the liquid medium to be assayed is come brined with various reagent compositions. Such compositions include a label conjugate comprising a binding component incur-prorated with a label. The binding component in the conjugate participates with other constituents, if any, of the reagent composition and the ligand in the medium under assay to form a Docket No. 2411-A -1 Lo ~3~09~8 binding reaction system producing two species or forms of the conjugate, erg., a bound-species (conjugate complex) and a free-species. In the bound-species, the binding component, e.g., a hasten or antigen, of the conjugate is bound by a corresponding binding partner, e.g., an antibody, whereas in the free-species, the binding component is not so bound. The relative amount or proportion of the conjugate that results in the bound-species compared to the free-species is a function of the presence (or -amount) of the ligand to be detected in the test sample.

Brief Description of the Prior Art -Many properties of natural cell membranes can be dipole-acted in simple lipid Baylor systems, referred to as lip-sores. One of these properties is louses. Louses can be achieved by conventional chemical agents, such as detergents, or by immunological reactions. when a Yesicu~ar t e.g., liposome, membrane contains an externally accessible antigen it will react with corresponding antibody, causing agglutination.
When the antigçn-sensitized liposome reacts with corresponding antibody in the presence of complement the membrane is ire versibly damaged and can no longer function as the intact selective permeability barrier This is immunolysisO The extent of imml~nolysis has been monitored by using antigen-sensitized liposomes containing entrapped marker molecules which are released upon immunolysis.

The louses of liposomes has been studied using a wide variety of marker systems. One type of marker system, which uses no marker-reactive reagents external to the liposome, has been disclosed in which the liposome encloses an electron pane-magnetic resonance spin marker, e.g., stable free radical, such as tempo choline. Tempo choline is water soluble but membrane impermeable, therefore it can be entrapped within liposomes or Docket No. 2411-A -2-23~
vehicles and it does not leak across the lipid bowlers.
Tempo choline enclosed within liposomes produces a character-fistic broad p~ramagnetic resonance signal of small amplitude.
This is because there is a high concentration of spin molecules which exchange signals due to their close proximity to one another. When the liposome membranes are ruptured r such as by activation of complement, the tempo choline molecules are released and diluted in the external medium. This results in a readily detectable, qualitative and quantitative, alteration in the paramagnetic resonance spectrum. Humphries, G. M. and McConnell, H. M. Pro. Nat. Aged. Sat. USA, 72: 2483-2487 tl9~). See, also, Wet, et at., J. Immunol. Methods, 9: 165-170 (1975); Chant et at., J. Immunol. Methods, 21: 185-195 (1978); and Asia, et at., Ann. NAY. Aged. Sat., 308: 139-148 (1978). Another marker system which uses no marker-reactive reagents external to the liposome employs a composition con-twining a flour, such as l-aminonaphthalene-3, 6, 8 trisulfon-aye, and a quencher, such as a, a'-dipyridinium p-xylene dibromide, entrapped within the liposome. Flour and quencher escape upon immunolysis ox the liposome and their subsequent dilution in the external volume abolishes the quenching, resulting in a high fluorescent signal. See Smolarsky, et at., J. Immunol. Methods, 15: 255-265 (1977) and Geiger, et at., Jo Immunol. Methods, 17: 7-19 (1977).

Another type of marker system employs an electrode in the reaction environment external to the liposome. One example ox this uses a potassium loaded liposome. Potassium ion escapes upon liposome immunolysis and reacts with an ion-selective electrode in the external environment. See Kowtows, et at., Chum.
Harm. Bull, 30: 1504-1507 (1982). Tetrapentylammonium ions have also been enclosed in liposomes and detected upon inn-louses using an ion-selective electrode See Schwab, et at., Anal. Chum., 52: 1610 (1980) and Chum. Let. 155 (1980). Also, so en erythrocyte ghosts (membranes) have been loaded with in-methylphenyl ammonium ion and louses has been detected with an Docket No. 2411-A -3-~L~31~fl~
ion-selective electrode. D'Orazio, et at., Anal. Chum., 49~
2083 (1977) and Croatia, et at., Anal. Chum. Act. 25: 109 ~1~79) One of the first types of marker systems to be reported employs substrate entrapped within the liposome or erythrocyte membrane vehicle. The substrate escapes upon immunolysis of the liposome and reacts with an enzyme-con~aining composition in the external volume to produce a detectable response. The early examples of this use glucose entrapped within the liposome. The glucose escapes upon immunolysis and its release from the liposomes is measured by the increase in absorbency at 340 nanometers which occurs upon reduction of NAP in the presence of hexokinase, glucose-6-phosphate dehydrogenase and the requisite cofactors. See Hixby, et at., Pro. Nat. Acadia Sat., 64: 290-295 (1969); Kinky, et at., Biochemistry, 8: 4149 4158 (1969); Kinky, en at., Biochemistry, 9: 1048 (1970)~ on a more recent example of this type of system, a fluorogenic substrate (umbelliferone phosphate) or a chromogenic substrate (p-ni'trophenyl phosphate) is enclosed in the liposome. The substrate escapes upon immunolysis of the liposome and reacts with an enzyme (alkaline phosphates) in the external volume.
Free substrate is produced, resulting in an increased signal.
See Six, et at., Biochemistry, 13: 4050 (1974); Uemura, et at., J. Become, 87: 1221 (1980); and Uemura, en at., J. ImMunol.
Methods, 53: 221-232 (1982).

Isis, including immunolysis, of -liposomes having internally entrapped enzymes as markers has also been reported.
The enzyme reacts with substrate in the external reaction medium resulting in a detectable response. For example, experiments have been performed using trapped enzyme markers including hexokinase, glucose-6-phosphate dehydrogenase and I-galactosidase. See ICataoka, et at., Become. Buffs. cat

2~8: 158-179 (1973). Also, horseradish peroxides can be entrapped within the liposome. The peroxides escapes upon Docket No. 2411-A -4-~31~4~
imm~nolysis and catalyzes the following reaction:

2 NASH 2 + 2 H -I 2 NOD t 2 H20 Oxygen is consumed by the oxidation of NASH and resultant pro-diction of water. The depletion of oxygen is detected by an oxygen electrode. See Hag, Become. Buffs. Rest Comma., go:
187-192 (1980).
-Several references report the enhancement of enzymicactivity upon louses of enzyme-containing liposomes. For example, Solomon, et at., Become. Buffs. Act, 455: 332-342 11976) report this observation upon louses of glucose oxidize-containing liposomes by either sonication or exposure to deter-gent. The enzymic activity of the entrapped glucose oxidize served as a measure for the permeability of the Baylor mom-brine of the liposomes to glucose in a non-separation assay The oxidation of-glucose was followed in the same reaction mix lure before and after detergent louses of the enzyme-containing liposome by oxygen uptake using an Ox electrode. Observed enzymic activity was as high as 4.60 times greater after louses than it was before louses.

Tweaking, et at., FOBS Letters, 106: 85-88 (1979) report an in vitro non-separation method for determining permeability of liposomes containing alkaline phosphates, a-glucosidase and a-galactosidase. Enzymic activity was determined using sub-striate and a coupled enzyme reaction in the same reaction mix-lure before and after detergent louses. Using alkaline phospha-tease, observed enzymic activity was as high as 17.5 times greater after louses than it was before louses. These authors state that if the Km of the particular enzyme substrate pair were the same both inside and outside of the liposome, the intravascular enzyme activity could be related to the apparent rate ox substrate permeability.

Docket No. 2411-A -5-I
McGee, et at., J. Cell Biology, 63: 492-504 (197~) have used horseradish peroxidase-containing liposomes. The enzymic activity of horseradish peroxides was determined Spectra-photometrically in the same reaction mixture before and after detergent louses. Ratios of observed enzymic activity before and after louses were as high as 8.3. They note that liposomes have been reported as useful models for membranes in permeably-fly studies owing to their sensitivity to pylon antibiotics and susceptibility to immune louses.

Several different homogeneous label conjugate specific binding assay systems are known in the art, one of which is disclosed by Pullman, et at., in U. S. Patent No. 4,193,983. In this assay system, a label and a ligand or ligand analog are nonequivalently bound to the external surface of a colloidal particle, such as a liposome, which is capable of maintaining its integrity in an aqueous environment. The discrete colloidal particle serves as a hub or nucleus which retain the ligand or its analog and the label in a substantially fixed average spatial relationship. By having the label and ligand in relatively close proximity on the surface of the particle, tube proximity of the label and the receptor bound to the ligand adjacent the label can be used in accordance with the prior art techniques to modulate the signal from the label. Nowhere is immunolysis suggested. Indeed, the clear teaching of this reference is that louses of the vehicle would adversely affect the spatial relationship proximity) of label and ligand which is required for this assay procedure.

Specific binding assay systems have been proposed, using a multilayered lipid membrane vehicle which has been prepared or treated to have surface-bound ligand or Lund analog and a marker or reagent substance enclosed within the vehicle. The remaining reagents for the assay include: (1) a binding partner, e.g., antibody, for the ligand; and (2) complement to effect louses of the vehicle upon binding of the binding partner to surface-bound ligand. generally, see McConnell, U. S.

Docket No. 2411-A 6-I
Patent No 3,850,578 and McConnell, et at., U. S. Patent No.

3,887,698 and Gregoriadis, et at., Liposomes in Biological So , John Wiley & Sons, N. Y. (1980), especially Chapter 12 entitled "Liposomes as Diagnostic Tools".

More particularly, immunoassay systems have been disk closed in which the use of enzyme-encapsulating liposomes is suggested. Asia, et at., U. S. Patent No. 4,235,792 describes a competitive homogeneous immunoassay method which employs immunolysis of an antigen-sensitized liposome containing a marker. Enzymes are among the markers disclosed (got. 6, lines 24-28).

Cole, U. S. Patent No. 4,342,826 discloses a specific binding assay using antigen-sensitized, enzyme-containing liposomes. These liposomes are immunospecifically caused to release enzyme upon binding of corresponding antibody and fixing of active complement. Upon enzyme release, the presence or absence of enzymatic activity is detected. Cole emphasize the advantage of providing a homogeneous system in which ~nzymic activity is substantially greater upon louses, e.g., a signal nose ratio of at least 5-10 and preferably above 60.

Each of the above approaches to vesicular marker systems has provided an advance of one tort or another in sequestering marker from the reaction medium and, thus, minimizing the generation of signal (latency) prior to immunolysis. That is, the signal observed from intact liposomes is considerably less than that from lucid liposomes. As demonstrated by the refer-encesr this end has been widely recognized as a major consider-anion in the improvement of liposome immunizes Further r the combined teaching of the literature in this area has been that the advantages to be achieved are enhanced when complete sequestration prior to louses is combined with the production of a high intensity signal upon immunolysis.

Docket No. 2411-A -7-I
SUMMARY OF THE Invention In contrast to the procedures previously described and in accordance with the present invention, it has been discovered thaw certain marker-containing liposomes generate a high intent sty signal while intact and a reduced or even extinguished signal (inverted latency) upon louses or alteration of membrane permeability. The present invention uses the combination of a vehicle with selected reagents, such that certain reagents out-side of the vehicle have access to the enclosed marker prior to any alteration of the vehicle and that alteration of the vehicle affords access by other reagents or components of the test composition. Advantages which can be achieved in accord-ante wit the present invention include increased sensitivity, at least to the range of about 10 18 molar (M) determinations of ligand in serum samples, reduction of serum interferences, simplicity of homogeneous specific binding assay protocols, and applicability to a broader range of high and low molecular weight ligands (analyzes) than previously possible for homage-nexus assays.

The above advantages are achieved by a test composition for determining a ligand in a sample, which composition coy-proses (a) a binding partner for the ligand; (b) a detection system which has at least two components; (c) a selectively accessible vehicle having a surface incorporated ligand or ligand analog and a first component of the detection system therein; (d) a substance which modifies vehicle accessibility in response to binding of surface-associated ligand or ligand analog and binding partner; and (e) at least one additional component of the detection system which is reactive with the first component to produce a detectable response which is reduced by association of the binding partner and vehicle mud lying substance with the vehicle.

Docket No. 2411-A -8-I
Further in accordance with the present invention, there is provided a specific binding assay method for determining a ligand in a sample, which method comprises combining the test composition of the invention with a sample suspected of con-twining ligand and observing a change in the detectable response. Preferably, the step of combining comprises stab-fishing a mixture of a sample suspected of containing ligand, a binding partner for said ligand, a luminescent reagent, couple mint and a ligand-sensitized, peroxidase-containing liposome;
incubating said mixture; and combining said mixture with hydra-gun peroxide. Luminescence of the mixture is observed after combination with hydrogen peroxide. The increase of intensity and duration of luminescent output is directly related Jo and results from the increased concentration of ligand in the sample. This increase in signal is associated with decreased availability of binding partner for binding to and initiation of complement fixation a the liposome surface. As noted above, this contrasts with signal responses previously observed where lower signal is observed with higher sample ligand con-cent ration.

As previously stayed, the liposomes of this invention exhibit higher luminescence when intact than when lucid. This phenomenon provides a basis for a useful and novel homogeneous immunoassay when such lyceums are sensitized by ligand or ligand analog. These can interact with specific binding partners, such as antibody, to provide a composition which will undergo louses in the presence of complement. Such louses leads Jo reduced luminescence. On the other hand, ligand in the sample competes with sensitized liposome for available antibody and, Jo the extent that such competition occurs, complement induced louses is reduced. Therefore, a dose-response curve can be constructed which relates increased luminescence intensity with increased concentration of ligand in the sample Docket No. 2411-A I

I

DESCRIPTION F TEE PREFERRED EMBODIMENTS

Preferred embodiments of the present invention include a luminescent specific binding assay reagent composition, a test device incorporated with one or more components of the test composition, a test kit comprised of containers or devices, each incorporated with one or more components of the test composition, in packaged combination with other components or materials, and methods of using the test composition, device and kit of the invention. . Specific terms in the following description which refer only to a particular embodiment are exemplary of all ox the embodiments unless otherwise indicated.
.

Sample fluids on which tests are performed include biological, physiological, industrial, environmental, and other types of liquids. Of particular interest are biological fluids such as serum, plasma, urine, cerebrospinal fluid.
saliva, milk, broth and other culture media and supernatants as well as fractions of any of them. Physiological fluids of interest include infusion solutions, buffers, preservative or antimicrobial solutions and the like. Industrial liquids include fermentation media and other processing liquids used, for example, in the manufacture of pharmaceuticals, dairy products and malt beverages. Other sources of sample fluid which are tested by conventional methods are contemplated as within the meaning of this term as used and can, likewise, be assayed in accordance with the invention.

The term "ligand" refers to any substance, or class of related substances, whose presence is to be qualitatively or quantitatively determined in a sample fluid, such as those just described. The present assay can be applied to the detection of ligands for which there is a specific binding partner and, conversely, to the detection of the capacity of a liquid median socket No. Lowe -10-to bind a ligand (usually due to the presence of a hinting partner for the ligand in the sample). The ligand usually is a peptize, protein, carbohydrate, glycoprotein, steroid, or other organic molecule for which a specific binding partner exists or can be provided by immunological or synthetic means.
The ligand, in functional terms, is usually selected from anti-guns and antibodies thereto; hastens and antibodies thereto;
and hormones, vitamins, metabolizes and pharmacological agents and their receptors and binding substances. Specific examples of ligands which can be detected using the present invention are hormones such as insulin, chorionic gonadotropin, thorax-ire; triiodothyronine, follicle-stimulating hormone, litanies-in hormone, thyroid-stimulating hormone, and easterly; anti-guns and hastens such as fourteen, bradykinin, prostaglandins, and tumor specific antigens; vitamins such as button, vitamin B12, folio acid, vitamin E, vitamin A, and ascorbic acid;
metabolizes such as 3', 5'-adenosine monophosphate and 3', I
guano sine monopho~phate; pharmacological agents or drugs sun as aminoglycoside antibiotics like gentamicin, amikacin an sisomicin, or drugs of abuse such as the opium alkaloids and ergot derivatives; antibodies such as microsomal antibody and antibodies to hepatitis and allergens; and specific binding receptors such as thyroxine binding globulin, avid in, intrinsic factor, and transcobalamin.

The terms "binding partner" or "receptor" refer to any substance, or class of substances, which has a specific binding affinity for the ligand in preference to other substances. In toe majority of embodiments, the present invention will incur prorate specific binding assay reagents which interact with the ligand or its binding effecters in the sample in an immune-chemical manner. That is, there will be an antigen-antibody or hapten-antibody relationship between reagents and/or the ligand or its binding effecter in the sample. Such assays therefore are termed immunoassay and the special interaction between the ligand and its receptor, or binding partner, is Docket No. 2411-A -11-immunochemical binding. Mohican antibodies are portico-laxly suitable receptors. However, it is well understood in the art that other binding interactions between the ligand and the binding partner serve as the basis of specific binding assays, including the binding interactions between hormones, vitamins, metabolizes, and pharmacological agents, and their respective receptors and binding substances. For example, palpated hormone receptors as binding agents or partners are discussed in Linguine, et at., (Ens.), Likened Assay, Mason Publishing U.S.A. In., New York, pages 211 et sex (1981).

The term "selectively accessible vehicle" refers to single or multi-compartmented sacs enclosing an internal volume, having a wall composed of one or more components and forming one or more internal compartments which constitute the internal volume. One example of such a vehicle is a cell ghost, wormed by opening a cellular membrane, removing the internal coupon-ens of the cell and resealing the membrane. Another example is a liposome, a single or multicompartmented vehicle comprised of lipids, particularly lipid mixtures including at least one phospholipid, which form a continuous wall or Baylor lipid membrane. A common constituent of these lipid mixtures it cholesterol. It has been observed that lipid mixtures Canaan in from about 10 to 40 percent (moles cholesterol/moles total lipid) can be used to prepare liposomes useful in the compost lion an method of tile invention iposomes can be prepared by any of a number of techniques For example, multilamellar vehicles (MLVs) can be prepared by film evaporation and hydra--lion of the lipid film. Reverse phase evaporation vehicles revs) may also be prepared. These are exemplary of techniques providing useful vehicles. For a general overview of liposomes and their formation, see Papahadjopoulos, et at., (Ens), Lip sores, Ann. NAY. Acadia Sat., volume 308 (1978); Tom, et at., weds.), , Elsevier North Holland Inc., NAY. (1980); and Gregoriadis, et at., Llposomes in Boo-logical Systems John Wiley & Sons, N Y. (1980).

Docket No. 2411-A -12-Liposomes can be made to have surface-incorporated ligand or ligand analog moieties. Such liposomes are formed using ligand-amphiphile conjugates, which usually take the form of a ligand~coupler-amphiphile molecule. Amphiphiles are sub-stances which contain both water soluble and water insoluble regions. They are best exemplified by the lipid amphiphiles, such as the phosphatidyl ethanolamines, phosphatidyl sonnet phosphatidyl instill, sphingomyelin cerebrosides, phosphate-die acid, plasmalogens, cardiolipins and fatty acids.

In coupling the antigenic material of interest to the amphiphilic molecule, a variety of coupling reagents and couple in reactions may be employed. Thus, for example, a carboxylic acid group of an antigenic material may be coupled to an amino group of an amphiphilic molecule by direct reaction to produce an antigenic N-substituted aside wherein the N-substituent is the residue of the amphiphilic molecule. In other examples r a distinct reagent is employed to couple the antigenic material to the amphiphilic molecule and this coupling reagent may be reacted initially with either the antigenic material or with the amphiphilic molecule to produce a reactive intermediate or a precursor which can thereafter be further reacted to produce the final ligand or sensitizer conjugate. Examples of such coupling reagents include azalea isocyanates, 4-fluoro-3-nitro-phenol asides, and malefic acid and derivatives which may be reacted with available amine groups to obtain N, N -subsidy maleimides. The tact that an antigen may first be coupled to a selected amphiphile, e.g., phosphatidyl-ethanolamine, -shrine or -instill and then included in the lipid mixture from which the liposomes are formed is most relevant inasmuch as this coupling reaction may be performed in a variety ox solvents which need not necessarily be compatible with other erg enzyme-containing, systems.

Alternatively, ligands may be covalently bonded or adsorbed to the surface of preformed liposomes. When liposo1nes are preformed, they can have at their external surface several Docket No. 2411-A -13-~31~)48 chemical functionalities to which antigens may be covalently linked. Foremost among these are: amino groups derived from phosphatidyl-ethanolamine, hydroxyl groups provided by phase phatidyl-inositol, and carboxyl groups provided by fatty acids or phosphatidyl-serine. Thus, antigens may be coupled to pro wormed liposomes by traditional chemical reactions - using bifunctional coupling agents such as: glutaraldehyde, doomed esters, aromatic and aliphatic diisocyanates, bis-p-nitro-phenol esters of dicarboxylic acids, aromatic disulfonyl color-ides and bifunctional arylhalides such as deflower-dini~robenzene; p,p'-difluoro-m,m'-dinitrodiphenyl cellophane Appropriate reactions which may be applied to such couplings are described in Williams et at., Methods_ n Immunoloqv_and Immunochemistry Vol. 1, Academic tress, New York (1967). I
some cases, antigens may be adsorbed to the liposome surface, as was shown by Uemura and Kinky, Biochemistry, 11:4085-4094 (1972).

The composition of the invention further includes a substance which modifies vehicle accessibility in response to binding of surface-incorpora~ed ligand or Lund analog and eye binding partner. The principal example of this substance is group of compounds collectively referred to as complement Complement is one of the main hum oral effecters of immune complex-lnduced tissue dBase through cell membrane disruption or louses. The binding of complement-fixing antibodies to ligands or ligand analogs on the surface of vehicles, such as liposomes or cell ghosts, has been shown to induce the fixation of complement. Resultant changes in membrane permeability, through actual membrane rupture or louses or formation of lung-tonal wholes" in the otherwise intact membrane, permit the release ox the detection system component(s) which had been within the vehicle or permit the access of components in the external medium to those detection components within the vehicle. The animal species from which complement is derived for-use should be compatible with the source of antibody aged antibody-sensitizing immunogen in accordance with known socket No. 2411-A -14-~31~
reactivities. For a general overview of complement and its effects, see Rasp, et at., Molecular Basis of Complement Action, Appleton-Century-Cro~ts (1970). Also, the role of complement is discussed in many of the references addressing other liposome immunoassay which have been cited above.

The composition uses a detection system which includes at least two components. The first component is within the vehicle and the at least one additional component is reactive therewith to produce a detectable response which is reduced by association of the binding partner and vehicle modifying substance with the vehicle. The principal example of this is a luminescent detection system. Luminescence is the chemical production of light. Analysis based on the measurement of emitted light offers several advantages over conventional techniques: high sensitivity, wide linear range, low cost per test, and relatively simple and inexpensive equipment. The types of luminescence that have engendered the most interest are chemiluminescence (CAL) and bioluminescence IBL)o The latter is the name given to a special Norm of luminescence found in biological systems, in which a catalytic protein increases the efficiency of the luminescent reaction Indeed, in certain cases the reaction is impossible without a protein component. See, in general, Crook, et at., (Ens.), Biochemical swoons, Marcel ~ekker, Inc., Nut"., NY (1~82);
Delco, et at., (Ens.), Bioluminescence and Chemiluminescence, Basic Chemistry and analytical Applications, Academic Press, NAY., NY (1~81); Mortise, et at., (Educe Luminescence from Biological and Synthetic Macromolecules, Eighth Katzir Conference, Ann. NAY. Aged. Sat., vol. 366 (1981); and __ Proceedings of International Symposium on Analytical Applique-lion of Bioluminescence and Chemiluminescence, State Printing Publishing, Inc. t West lake Village, CA (1979)~

The process of CAL in solution involves three stages: (a) preliminary reactions to provide the key intermediate; (b) an Docket No. 2411-A -15-excitation step in which the chemical energy of the key inter mediate is converted into electronic excitation energy; and (c) emission of light from the excited product formed in the comma-eel reaction. In general, the reactants include an oxidant and reluctant where the reluctant, as a consequence of briny oxidized, emits light. Often, it is preferred to include a catalyst such as home or peroxides. The CAL reactions desk cried in this section all have the common feature of detecting hydrogen peroxide, an oxidant. This makes them of particular interest to the clinical chemist because they offer an alte.na-live to the calorimetric peroxide detection methods now used in many clinical assays.

In general, hydrogen peroxide is the most commonly used oxidant. Other oxidants used include ethyl hydroperoxide, hypochlorite, iodine, permanganate, and oxygen in the presence ox a suitable catalyst. Enzyme systems, including coupled enzyme systems, which produce oxidants such as hydrogen Xerox-ire as an intermediate or end product have been used as a source of such oxidant for the luminescent detection system in accordance with the invention. Examples are the glucose/glu-cove oxidize or glycerol/glycerol oxidize reactions to produce hydrogen peroxide.

The phenomenon of chemiluminescence as a consequence of reaction with hydrogen peroxide has been found in several classes of reluctant compounds which emit light (luminophores)f particularly the cyclic diacylhydrazides. One of the most commonly used is luminol (5-amino-2,3-dihydrophthalazine~
drone). A shift in the position of the amino group reduces efficiency, e.g., isoluminol (6-amino-2,3-dihydroph~halaæine-Dunn) is 10% as efficient as luminol. Substitution in the ring structure markedly influences the luminescence. Electron-withdrawing substituents in the Bunsen ring decrease luminous pence, but electron-donating substituents increase light yield substitution at positions 5 and 8 being more effective Docket No 2411-A -16-3~8 than at 6 and 7. A complete loss of light occurs if the hotter-cyclic ring is substituted. Annotated analogs of luminol have been- produced that are 300% more efficient than luminol. See, McCapra, the Chemiluminescence of Organic Compounds, Rev London), 20:485 (1966). Several other chemiluminescent systems have been developed including those which use lucigenins (e.g., bis~N-methylacridinium nitrate), acridinium pinwheel carboxylates, diary oxalates such as bus (trichloro-phenol) oxalate, lophine and polyhydric phenols such as pyre-gallop or garlic acid. Excellent reviews on luminescence and its role in chemical assays are provided in Gurus, et at., Applications of Boo- and Chemiluminescence in the Clinical Laboratory, Olin. Chum., Z5:512-519 (197g) and in Whetted et at., Ana_x~_cal_ Luminescence: Its Potential in the Clinical laboratory, Olin. Chum. 25:1531-1546 (1979).

Bioluminescence (BY), as noted above, is a form of luminescence found in biological systems. In most BY systems, the enzyme luciferase catalyzes the luminescent oxidation of a substrate, Lucifer in. The generic term "luciferase" refers Jo on enzyme that catalyzes the oxidation of a substrate, such as Lucerne, to produce light. The generic term "Lucifer in"
refers to a reduced compound that can be oxidized to an electronically excited singlet state and which produces light upon its return to the orotund state. The mast completely studied of these systems is that of the firefly, or which the reaction is illustrated as follows:

Lucifer in AT + 2 luclf~2as~ ASP COY fight This reaction is best carried out at about 25C in Gleason buffer (pi 7.8). Most of the remaining AL systems have been observed in marine organisms, such as marine bacteria. For example, the reaction system in Vibrio flscherl and eons Harvey is illustrated as follows:

Docket No. 2411-A -17-~3~8 NASH FUN reeducate FMNH2 + NOD
FMNH2 + RHO 2 l die as FUN + RC02~ fight In this reaction RHO is a long chain aliphatic alluded. The reaction is best carried out at below 25C in a pi range of about 6.4 - 7.2 for V. Fischer and pi 5.6 ~6.8 for By Harvey.
these and other BY systems are also fully discussed in Gurus, et at., swooper and Whetted, et at, swooper.

The term "peroxidatively active substance" defines the precise chemical activity of the substances contemplated. A
peroxides is an enzyme which will catalyze a reaction wherein hydrogen peroxide oxidizes another substance. The peroxidases are generally proteins which incorporate iron porphyrin moieties. Peroxides occurs in horseradish, potatoes, fig tree sap and turnips (plant peroxides); in milk (Luke peroxides), and in white blood corpuscles overdo peroxides); also it occurs in microorganisms and may be produced by fermentation.
Certain synthetic peroxidases, such as those disclosed by Thrill and Meekly in Act Chum. Stand., 4:422-434 (1950), are also satisfactory for use in 2 detention systems. Less sat-is factory are such substances as hymen, met hemoglobin, oxyhem2 glob in, hemoglobin, hemochromogen, alkaline Hampton, hymen derivatives, and certain other compounds which demonstrate peroxidative or peroxidase-like activity, namely, the ability to catalyze the oxidation of another substance by means of hydrogen peroxide and other peroxides. Other substances which are not enzymes but which demonstrate peroxidati~-e activity are: iron sulfocyanate, iron twenty, ferrous ferrocyanide, chronic salts such as potassium chronic sulfate) absorbed on silica gel, etc.

As noted above, the luminescent detection produces light energy. It is possible to transfer this energy to another molecule(s) which produces a detectable response a a different wavelength or of a different type altogether. For example a Docket No. 2411-A -18-flour can be added to the detec~lon1 stem. The luminophore and flour can be selected such that light is emitted at the excitation wavelength of the flour. Fluorescence is then detected at the wavelength dictated by the flour selected.
This and other energy transfer mechanisms can be combined with the luminescent system to modify the detectable signal as desired. One advantage of energy transfer systems is to over-come or avoid the interference of endogenous or background emissions of the sample under assay which are not readily disk tinguishable from the signal produced by the luminescent system.

Also, various inhibitors, quenchers or modulators can be included to modify the reactivity or signal of the detectioll system. Substances which inhibit or diminish enzyme activity are known and can be incorporated to affect the enzyme upon its release from the liposome or to penetrate this liposome upon modification of the permeability. For example, cyanide ions have been observed to reduce the activity of released porks-dBase without otherwise affecting the assay system. Also, anti-bodies have been described which inhibit peroxides activity.
substances which quench luminescent or fluorescent signals can be incorporated to enhance the inverted latency phenomenon observed. Several such quenching substances and mechanisms are known For example, the above-described luminescence/fluores-pence energy transfer can be accomplished using a fluoresce whose fluorescence is quenched when antigen/antibody binding has occurred. This has been described with respect to Canaan-tonal binding assay systems in J. Olin. Path., 30:526 ~1977).
Enzyme modulator systems have been described in Boguslaski, et at., US. Patent No. 4,134,792.

The method of assay in accordance with the invention normally involves the combination of ligand-containing sample t antibody specific for the ligand sensitized, peroxides-containing liposome and complement. After a suitable incubi-lion period the above mixture is combined with the luminescent Docket No. 2411-A -19-compound and hydrogen peroxide a eye o~dant. The lupines-pence thereby generated is determined with a light sensitive detector such as a photo multiplier tube, photographic film or semi conductive diode.

The following working examples describe experiments which were performed in developing the present invention. Standard commercially available reagent grade chemicals were used whenever possible.

Docket No. 2411-A -20-The experiments reported by this example demonstrate the change in luminescence of a reaction mixture comprising a horseradish peroxidase-containing liposome (HRP/liposome), a chemiluminescent (CAL) compound and an oxidizing agent for the CAL compound when the mixture is exposed to a detergent which lyres the liposomes~ A most unexpected phenomenon was observed as described below.

Horseradish peroxidase-containing liposomes were prepared as follows. A lipid film was formed by evaporation of a mix-lure of 13 milligrams (my) egg lecithin (Sigma Chemical Co., St. Louis, MO); 3 my dustily phosphate (Sigma Chemical Co., swooper); and 1.1 my cholesterol in 12 ml of 4:1 sheller-.

form/methanol, volume Jo volume (v/v), on the inside surface ova pear shaped flask. Evaporation was conducted under vacuum my water aspiration at centigrade (C) until a dry film was observed. Thereafter, this film was further dried for two hours under vacuum at 0.5 mm mercury. The lipid film so formed was hydrated with 4.0 ml of TRIP buffer containing 200 my horseradish peroxides (HOP) (Miles Laboratories, Inc., Elk hart, IN) in each ml of TRIP buffer overnight at 4C with stirring by a magnetic bar. The iris (hydroxymethyl) amino-methane (TRIP) buffer stock solution (TRIP buffer) was made by combining 100 ml of a TRIP solution (60.5 g/l; pi 8.6) with 150 ml of an Nail solution (58.5 9/1) and adjusting the volume to 1.0 liter with distilled water to give final concentrations of 0.05 molar (M) TRIP and 0.15 M Nail. The liposomes so formed were separated from free peroxides by gel exclusion chrome-tography of a 1.0 ml volume of the lipid suspension on a 1.5 x 32 centimeter (cm) column of Suffers 6B (Pharmacia Fine Chemicals, Pussycat, NJ) using TRIP buffer as the fluent.

Docket Noah -21-* Trade Mark ~23~
Forty effluent fractions of 1.8 ml were collected. ~RP/lipo-sores were isolated in fractions 11-14 as confirmed by the presence of an absorbency peak (due to turbidity) at 410 no using a Gil ford Model 250 spectrophotometer (Gil ford Incitory-mints, Inc. Oberlin, OH. Fractions 12 and 13 were combined and stored under a nitrogen atmosphere at 4C.

Luminol reagent solution was prepared as follows. A 221.5 my portion of luminol (Aldrich Chemical Co., Milwaukee, WI) was dissolved in 50 ml of TRIP buffer, to which 4 drops of 50% Noah (w/v) were added to completely dissolve the luminol, producing a 2.5 x 10 EM luminol solution. A 0.5 ml volume of this soul-lion was mixed with 49.5 ml TRIP buffer to provide the luminol reagent solution having a 2.5 x 10 M l~minol concentration.

The HOWE reagent was made by adding 14 I of 30% (v/v) aqueous HOWE (Fisher Scientific, Orange burg, NY) to 50 ml TRIP
buffer.

The combined HRP/liposome fraction (fractions 12 and 13) was assayed by catalytic oxidation of luminol in the presence of HOWE as follows. A 1.0 ml volume of each of a series of dilutions of ~RP/liposomes was prepared in TRIP buffer. An equivalent series of dilutions was prepared in TRIP buffer con-twining 1% wove) TRITON-X-100 (Room & Hays Co., Philadelphia, PA). A 100 I volume of each dilution was injected into a separate polypropylene tube which was positioned in a Turner Model 20 Luminometer (Turner Designs, Mountain View, CA) and which contained 200 I of 2.5x10 4 M luminol and 200 I of 2.5x10 3 M HOWE in TRIP buffer. After the reaction was thus initiated in the particular reaction mixture being assayed r luminescence was monitored in each by integration of the signal for 60 seconds after a 0.5 second delay and recorded as Aruba-tray luminescence units. The observed integrated luminescence for ~RP/liposomes chemically lucid with TRITON-X-100 and for Docket No. 2411-A -22-* Trade Mark intact HRP/liposomes, as well as the ratio of the signal from lucid and intact HRP/liposomes (L/I) fur each of the liposome dilutions are set forth in Table I.

TABLE I

~RP/liposomes CAL Response CAL Response (dilutions) (with Briton) _ no Triton1 L/I Ratio 1/5 140 670 0.21 1/10 18.~ 324 0.0~
1/50 1.5 I OWE
1/100 0.7 30 0.02 1/500 0.24 6.2 0.04 . 0 Old 0.29 0~66 The data in Table I document an unexpected phenomenon.
The luminescence observed for intact liposomes is greater than what for chemically lucid liposomes. This represents an inverse latency in that the luminescent response of lucid lip-sores divided by the luminescent response of intact liposomes is less than 1.0~ This observation represents the basis for construction of a specific binding assay for which the lupines-pence derived from intact liposomes is greater than the lump-nascence derived from liposomes which are lucid by antibody and complement, as demonstrated in later Examples.

Docket No. 2411-A -23-* Trade Mark I`

EXAMPLE II

The next experiments which were performed investigated whether the greater luminescence of intact liposomes over the quenched" luminescence of chemically lucid liposomes, as observed and reported in Example I, was caused by exposure and potential inactivation of free peroxides by the louses agent, e.g., TRITON-X-100 detergent.

To begin, solutions of HOP were prepared by adding 2.0 my of RIP to a OWE ml Alcott of TRIP buffer giving a concentra-lion of 1 x 10 5 M HOP, as determined by the absorbency at 403 no using an extinction coefficient of 91mM 1 cm 1. A series of dilutions of this HOP solution was prepared in TRIP buffer. An equivalent series of dilutions was prepared in TRIP buffer con-twining 1% (w/v) TRITON-X-100~ A 100 I volume from each of these solutions was injected into separate 8 x 50 millimeter (mm) polypropylene tubes which were positioned in a Turner Model 20 luminometer and which contained 200 us of 2.5 x 10 4 M
luminol and 200 us of 205 x 10 3 M HOWE in TRIP buffer. After the reaction was thus initiated in the particular reaction mix-sure being assayed, luminescence was monitored in each by into-gyration of the signal for 60 seconds after a 0.5 second delay and recorded. The observed integrated chemiluminescence for HOP solutions with and without TRITON-X-100, as well as the ratio for each of the dilutions is set forth in Table II.

TABLE II
CAL Response CAL Response [HOP] M (with TRITELY (no BRITON) Ratio 2 x 10-7 1886 1598 1.18 5 x 10-8 355 295 1.20

4 x 10-8 247 20~ 1.22 2.5 x 10-8 126 101 1.25 0 0~1 0.1 1.00 Docket No. 2411-A -24-* Trade Mark I

Enhancement of peroxides catalyzed oxidation of luminol by H202 was observed in the presence of TRITON-X-100. Thus, TRITON-X-10~ does not inhibit peroxides catalyzed oxidation of luminol and is not the cause of the quenching phenomenon reported in the previous Example.

Docket No. 2411-A -US-* Trade Mark I
EXAMPLE .

The next experiments which were performed investigated the effect of empty liposomes on the chemiluminescent signal observed in a free peroxidase-catalyzed oxidation of luminol by HOWE. This was done to determine whether the inverted latency phenomenon would result from the combined presence of free peroxides and empty liposomes.

Liposomes were prepared as follows. First, a lipid film was formed by evaporation of a mixture of 7.3 my egg lecithin, 1.75 my dozily phosphate and 2.9 my cholesterol in 13 ml of 4:1 C~C13/methanol (v/v) to dryness on the inside surface of a ml pear-shaped flask. Evaporation was conducted under vacuum as described in Example I. The lipid film so formed was hydrated with 2.0 ml of TRIP buffer, to which no peroxides had been added, overnight at 4C with stirring by a magnetic stirring bar and the liposome preparation so formed was maintained by storage a 4C for use in the manner described below.

Jan HOP solution was prepared by adding 13.6 my of RIP is

5.0 ml of TRIP buffer giving a concentration of 5 x 10 5 M HOP, as determined by the absorbency at 403 no using an extinction coefficient of 91mM 1 cm 1. This solution was then diluted to a concentration of 5 x 10 7 M by combining 0.25 ml with 25 ml TRIP buffer to make a solution which was used to prepare samples with if) enzyme only, (2) enzyme plus TRITON-X-100, (3) enzyme plus empty liposomes, and (4) enzyme plus TRYOUT and empty liposomes. The formulation of each of the above mixtures and controls (minus HOP) is set forth in Table III. A 100 I
volume of each of these solutions was injected into separate polypropylene tubes which contained 200 I of 2,5 x 10 4 M
luminol and 200 ye of 2.5 x 10 3 M ~22 in TRIP buffer and were Docket No. 2411-A -26-* Trade Mark , I
positioned in a Turner Model 20 Luminometer. After the react lion was thus initiated in the particular reaction mixture being assayed, luminescence was monitored by integration of the signal for 60 seconds after a 0.5 second delay and recorded.
The CAL response was observed in each tube in the same manner as described in Example I and is also set forth in Table III.

TABLE III
_ ___ HRPTRIS Buffer I BRITON Liposomes CAL
_ I I us I Response (a) 0 1000 0 0 2.7 (b) 0 300 700 0 1.9 (c) 0 800 0 200 0.8 (d) 0 100 700 200 0.6 Tao 950 0 0 5.1 (f~50 250 ? o 4.2 (g)50 750 0 200 2.0 (h)50 50 700 200 2.6 (i) 100 900 0 0 11.3 (j) 100 200 700 0 12.3 (k) 100 700 200 5.1 (1) 100 0 700 200 7.2 The data in Table III first shows that only very slight luminescence is observed in preparations I which contain-Ed no RIP particularly in preparations (c) and (d) which included empty liposomes. Preparations I show high level luminescence in the presence of 50 I RIP In contrast, preparations (g) and (h) contained 50 I RIP and empty lip-sores in the absence and presence, respectively of TRITON-X-Andy displayed a luminescence which was only half that seen in the absence of liposomesl A comparison of preparations (g) and (h) shows that TRITON-X-100 has the effect of slightly increasing luminescence in the presence of liposomes. Prepare-lions (i) and (j) show high level luminescence in the presence of 100 I HOP. In contrast, preparations (k) and (1) contained Docket No. 2411-A 27-* Trade Mark I
100 us HOP and empty liposomes in the absence and presence, respectively, of TRITOr~-X-100 and displayed substantially less luminescence. comparison of preparations (k) and (1) shows that TRITON-X-100 had the effect of slightly increasing luminescence in the presence of liposomes. In summary, addition of empty liposomes to horseradish peroxides external to the liposome resulted in decreasing the luminescent signal.
When ~RITON-X-100 was included in the incubation mixture the chemiluminescent signal was enhanced.

Docket No. 2411-A -28-* Trade Mark I, . .. .. .

EXAMPLE IV

LUMINESCENT LTPOSOME THEOPHYLLINE ASSAY

Theophylline (1,3-dimethylxanthine) is commonly used in the treatment of bronchial asthma. The serum concentration of the drug must be closely monitored since the drug has a narrow therapeutic range of 10-20 gel while drug concentratiorls in excess of 20 Jyg/ml may be toxic. The experiments reported below demonstrate that the inverted latency luminescent immunoassay of the invention can be used to quantitate serum levels of theophylline.

Sensitized HRP~Liposome Preparation Theophylline-sensitized HRP/liposomes were prepared as follows. First, a lipid film was formed by evaporation, as described in Example I, of a mixture ox 7.3 my egg lecithin, 1.7 my dustily phosphate, 0.6 my cholesterol and 0.15 my theophylLine conjugate in 10.0 ml of 4:1 chloroorm/methanol (v/v) on the inside surface of a pear-shaped flask. The theophylline sensitizer used was a theophylline-dipalmitoyl phosphatidyl ethanol amine conjugate (theophylline-DPPE). Such a conjugate can be prepared by the procedure described in Elaga, et at., Become. Biophvs. Res._Comm., 95: 187-192 (1980). This same theophylline-DPPE conjugate was synthesized by a modified procedure and analyzed to confirm its identity and purity. the purified conjugate was obtained as a preparative liquid chrome-tography isolate which showed a single spot on thin-layer chromatography. The identity of this conjugate was confirmed by MY W and IT spectra and elemental analysis.

Docket No. 2411-A -29-I
The lipid film so formed was hydrated with 2~0 ml of TRIP
buffer containing 2 mg~ml HOP overnight at 4C with stirring by a magnetic stirring bar. The theophylline-sensitized ~RP/lipo-sores so formed were separated from free peroxides by chrome-tography of 1.0 ml of the lipid suspension on a 1.5 x 32 cm column of Suffers 6B using TRIP buffer as the fluent. Forty effluent fractions of 1.8 ml were collected. Theophylline-sensitized ~RP/liposomes were found in fractions 12-14 and free peroxides in fractions 27-34.

Each of the effluent fractions so collected were assayed spectrophotometrically to confirm the presence or absence of peroxides as follows. A 50 pi Alcott of each fraction was combined with 200 I of TRIP buffer. Each of a parallel set of fraction allocates was combined with 200 I of TRIP buffer which contained 1% (w/v) TRITON-X-100. A 20 I alto of each preparation was introduced into a 1.0 cm optical path length cuvette which contained 3.0 ml TRIP buffer and 50 I of a stock gawkily (O-methoxyphenol; Mathewson, Coleman & Bell, Nerd, OH) reagent (2.45 mg/ml TRIP buffer). Each of these cuvettes was positioned in a Gil ford Model 250 spectrophotometer and the reaction was initiated by addition of 50 I of an ROY stock solution, made by combining 50 us of 30% (v/v) aqueous HOWE
solution (Fisher Scientific Co., Pittsburgh, PA) with 100 ml TRIP buffer. The contents of each cuvette were monitored at 436 no wavelength for 60 seconds after initiation of the reaction. The results of this procedure confirmed the presence of peroxides in fractions 12-14 and 27 to 34. Since liposomes were observed, as noted above, only in fractions 12-14, it was ascertained that fractions 27 to 34 contained free horseradish peroxides.

Docket No. 2411-A -30-* Trade Mark ..~, . I, . ' I .

I
Other Runt Theophylline samples of 1.25, 2.5, 5, 10, 20, 50 and 100 ug~ml theophylline were prepared from a stock theophylline solution, which consisted of 5.3 my theophylline (Sigma, dissolved in 50 ml TRIP buffer, by appropriate dilution of allocates with-additional TRIP buffer.

Anti-theophylline antibody reagent was prepared from a commercially available anti-theophylline rabbit antiserum (Kallestad Inc., Austin TX, Catalog No. 334) as follows. A
100 us volume of the Kallestad antiserum was added to 900 us of TRIP buffer to make a 1:10 (v/v) stock antibody solution.

Complement reagent was prepared as follows. A vial con-twining lyophylized guinea pig serum (Pot Freeze, Rogers, OK, Catalog No. 38005-1, was reconstituted with 3.0 ml distilled water. A 0~5 ml volume of the reconstituted complement-containing serum was combined with azide-TRIS buffer to make a 1:10 (v/v) dilution, used in the assay procedure. This aside TRIP buffer was made by dissolving 208.1 my sodium aside in 100 ml TRIP buffer.

Assay Procedure and Results .. . _ . . . .

First a 10 I volume of each of the theophylline samples described above was dispensed into one tube of two duplicate sets of test tubes. Then 50 us of a 1:5 dilution in TRIP buffer of the stock antibody solution was introduced into each tune of the first set of tubes and 50 ye of a 1:10 dilution of stock antibody in TRIP buffer was introduced into each tube of the second set. Next, 40 I of a 1:4 dilution in TRIP buffer ox the theophylline-sensitized liposome preparation (fraction 13) was introduced into the mixture in each tube of the first set and Docket No. 2411-A -31-I
40 I of a 1:10 dilution in TRIP buffer was introduced into the mixture in each tube of the second set. Both sets were then incubated at 37C for five minutes. After the five minute - incubation period, a 100 us volume of a 1:10 dilution in TRIP
buffer of the complement reagent was introduced into the mix-lure in each tube of both sets. They were then incubated at 37& for an additional five minutes.

Controls were also prepared using 10 I volumes of a 10, 50, 100 ug/ml theophylline sample and a blank TRIP buffer which contained no theophylline by adding 40 I of the 1:4 dilution ox the liposome preparation and 150 us of TRIP buffer in lieu ox the antibody and complement. A 100 I Alcott of each of these preparations was withdrawn and injected into a separate polypropylene tube positioned in a Turner Model 20 Luminometer and which contained 200 us of the 2.5 x 10 4 M luminol reagent and jowl of the 2.5 x 10 3 M H202 reagent. After the Rex was thus initiated in the particular reaction mixture being assayed, luminescent output was monitored by integration for 60 seconds aster a 0.5 second delay and recorded. The results observed are set worth in Table IV.

CAL Response Theophylline 1/4 dilution 1/10 dilution Control of liposomes of_liPosomes mixture 0 157 55.0 So 1.25 139 57.5 2.5 181 63.6 186 98.5 274 125.5 272 126.2 266 100 256 114.7 257 Docket No. 2411-A -32-~Z3~
- An increase in luminescence is observed with increasing sample theopkylline concentration for both reagent formula-lions. In contrast, the control solutions produced uniformly undiminished luminescence, regardless of sample theophylline concentration. This clearly and simply demonstrates the apply-cation of the inverted latency phenomenon to an immunoassay composition and method for the quantitative determination of theophylline levels at least as low as 1.25 gel and at con-cent rations 5-fold that of the threshold toxic dose.

- octet No. 2411-A -33-EXAMPLE V ~3~4~

This example demonstrates the effect of an inhibitor of peroxides, cyanide ion, on the signal observed for intact liposomes and liposomes which were chemically lucid with TRITON-X-100 in a luminescent assay for peroxides in which luminescence of the reaction mixture is recorded at a fixed time interval after initiation of the reaction.

Liposomes prepared in Example I were separated from free peroxides by gel exclusion chromatography by the procedures described in Example I. Effluent liposome fractions were come brined and stored as a stock suspension at 4 degrees C. A work-in suspension of a 1/40 dilution of the stock liposome suspend soon was prepared in TRIP buffer (intact liposomes). Semi-laxly, a 1/40 dilution of the stock liposome suspension was prepared in THIS buffer containing 0.2% (w/v) TRITON-X-100 (lucid liposomes).

A 1 my stock solution of sodium cyanide (Nan) in TRIP
buffer (1 my CN-TRIS) was prepared by dissolving 5.0 my of Nan in 100 ml of TRIP buffer. Reagent solutions of sodium cyanide in TRIP buffer were made from 1 my CN-TRIS to contain 0.083, 0.2, 0.33, 0.5 and 0.75 my Nan in TRIP buffer.

Luminescence of intact and chemically lucid liposomes was measured by injection of 100 I of thy 1/40 liposome dilution into separate polypropylene tubes which contained 300 us of either TRIP buffer or 300 ye of one of the cyanide reagent solutions, 50 us of SX10 3 M HOWE in TRIP buffer, and 50 pi of M luminol in TRIP buffer. These tubes were positioned in a Turner Model 20 luminometer. Luminescence was recorded at 15 minutes after initiation of the reaction. Table V sets forth the observed luminescence for intact and chemically lucid lip-sores, as well as the ratio of the signal from lucid and intact Docket No. 2411-A -34-* Trade Mark I

;23~
HRP/limpo~omes, (L/I ratio), for each of the concentrations of cyanide, [ON] my, in the reagents.

TABLE V

CAL RESPONSE CAL RESPONSE
[ON] EM WIT BRITON NO BRITON LOWE RATIO
r _ 0 1056 1388 0~76 0~083 10~9 927 0~01 0~20 17~3 913 0~02 0~33 27~0 963 ~03 0~50 27~9 997 0~03 0~75 30.1 1004 0~03 1~00 21~9 921 0~02 Under conditions of the experiment in which no cyanide was present, the L/I ratio was 0. 76~ At the lowest concentration cyanide reagent, 0~083 my, the L/I ratio was 0.01. Lupines-pence of intact liposomes was inhibited no more than 34% by the range of cyanide concentrations tested. Luminescence of chemically lucid liposomes was inhibited more than 97% at con-cent rations of cyanide used in these experiments. Thus, this demonstrates that the presence of cyanide enhances the inverted latency phenomenon observed.

Docket No. 2411-A -35-* Trade Mark EXAMPLE VI ~3~048 This example demonstrates an inverse latent ratio by use of an inhibitor of peroxides, cyanide ion, under conditions such that an inverse latent ratio is not otherwise observed.

The theophylline sensitized liposomes containing horse-radish peroxides prepared in Example IV were used in this series of experiments. A working suspension of liposomes was prepared by diluting an Alcott of fraction 13 into TRIP buffer as follows. A 12 I Alcott of fraction 13 was added to 588 us of TRIP buffer to make a 1/50 dilution (intact liposomes).
Similarly, a 12 I Alcott of fraction 13 was added to 588 I of TRIP buffer containing 0.7% (w/v) TRITON-X-100 to make a 1/50 dilution lucid liposomes).
.
A reagent solution of Owe my sodium cyanide in TRIP
buffer was prepared by addition of 2.0 my of 1 my CN-TRIS to 4.0 my of TRIP buffer.

Luminol reagents were made from a stock solution of 2.5 x 10 2 M luminol in TRIP buffer prepared as described in Example I by dilution into TRIP buffer. Concentrations of luminol reagents are set forth in Table VI.

hydrogen peroxide reagents were made as follows. A stock solution of 5 x 10 2 M ~22 was prepared my addition of 57 I of 30% (v/v) aqueous H202 to 10.0 ml of TRIP buffer. Next, this stock solution was used to prepare the hydrogen peroxide reagents by dilution with TRIP buffer to give the noncentral lions set forth in Table VI.

Luminescence of intact and chemically lucid liposomes was measured by injecting 100 I of the working suspension of intact liposomes or 100 I of the working solution of comma-calmly lucid liposomes into separate polypropylene tubes which Docket No. 2411-A -36-* Trade Mark .
`'' ''' ' 23~
contained 300 us of 0.33 my CN-TRIS, 50 jut of one of the luminol reagents and 50 I of one of the HOWE reagents. The tubes were positioned in a Turner Model 20 luminometer. Lump-nascence was monitored for the reaction in each tube by into ration of the signal for 60 seconds after a 0.5 second delay and recorded.

Luminescence was also recorded for intact and chemically lucid liposomes in a control experiment for which the cyanide reagent was replaced with TRIP buffer. The observed lupines-pence, concentration of luminol and hydrogen peroxide in the reagents and the ratio of the signal or lysed/intact loupe sores, (L/I ratio), without cyanide is set forth in Table VI, experiment (a). Similarly the observed luminescence for experiments in which the concentration of sodium cyanide in the reaction mixture was 0.2 my is set forth in Table VI, export-mints be TABLE VI

[~22] [LUMINOL] CAL RESPONSE CAL RESPONSE
M M WIT BRITON NO BRITON L/I RATIO
(a) 10-3 owe 630 112 5.6 (b) 10 3_2 owe 5.8 67 0.09 (c) 5x10 10 2 6.0 736 0.008 Ed) 10 2 owe 11.0 456 0.02 (e) 10 2 10-3 71 0.05 he data in Table VI show that under conditions where an inverse latent ratio is not observed, experiment (a), addition of an inhibitor of peroxides to the luminescent reaction mix-lure, experiment (b), results in diminution of the signal from lucid liposomes to a greater extent than for intact liposomes4 The L/I ratio was 5.6 with no cyanide in the reaction mixture, experiment (a), while the L/I ratio was 0.09 in the presence of Docket No. 2411-A -37-* Trade Marc I

I
an inhibitor of peroxides, cyanide, experiment (b). Export-mints I show the inverted latency over a range of H202 and luminol concentrations.

Doclcet No 2411-A -38-EXAMPLE VII

This example describes the use of liposomes containirlg horseradish peroxides in an immunoassay procedure for the-felon in which the luminescent signal derived from intact liposomes is greater than that of lucid liposomes. Peroxides was assayed by oxidation of luminol with ~22 as the oxidant.
Luminescence was monitored by recording the signal at a fixed time after initiation of the reaction. An inhibitor of porks-dBase, cyanide ion, was included in the luminescent reaction mixture.

event Preparation The theophylline-sensitized liposomes containing porks-dBase prepared in Example IV were used for this series of experiments. A liposome suspension was prepared by dilution of fraction 13, Example IV, 1/10 with TRIP buffer (liposome reagent).

Anti-theophylline antiserum and complement were from the same source described in Example IV. Antibody reagent was pro-pared by dilution of anti-theophylline antiserum 1/10 with TRIP
buffer (antibody reagent). Complement reagent was prepared by ~i1ution of guinea pig serum 1/10 with TRIP buffer (complement reagent).

Theophylline samples of 2.5, 5, 10, 20, 40, 60, and 100 gel were prepared from the stock theophylline solution of Example IV, by dilution with TRIP buffer.

A reagent solution of 5xlO 2 M HOWE was prepared by add-lion of 57 I of 30~ HOWE (v/v) to 10.0 ml of TRIP buffer (HOWE
reagent).

Docket No. 2411-A -39-I
A reagent solution o 10 2 M luminol was prepared by disk solving 179 my of luminol in about 80 ml of TRIP buffer to which 4 drops ox 50% Noah (w/v) were aided. The pi of this solution was adjusted to I with dilute Hal and the volume adjusted to 100 ml by addition of TRIP suffer (luminol reagent).

A reagent solution of 0.33 my Nan in TRIP ~ufferl pi 805 was prepared as described in Example VI (cyanide reagent).

Assay Procedure and Results Y . ._ In separate jest tubes, 20 us of one of the theophylline samples as described above, or 20 I TRIP (O gel theophyll-ire) was incubated with 50 I of antibody reagent and 100 I of complement reagent at 37 degrees C for 5 minutes. Liposome reagent, 50 I was added to the mixture of sample, antibody and complement reagent and incubation was continued at 37 degrees C for another 2 minutes A lOOJul Alcott from each of these assay mixtures was withdrawn and injected into separate polypropylene tubes positioned in a Turner Model 20 Lyman meter. Each polypropylene tube container 300 I cyanide reagent, 50 I ~22 reagent, and 50 I luminol reagent.
Luminescence was recorded 11 minutes after initiation of one reaction in each tube.

The concentration of theophylline in each sample and the observed luminescence is set forth in Table VII.

Docket No. 2411-A -40-TABLE VI I
I
[THEOPHYLLINE]
gel LUMINESCENCE
.
O ;~65 2. 5 341 A dose response relationship between the luminescence of the mixture of sample, antibody reagent, complement reagent liposome reagent, cyanide reagent, H202 reagent and luminol reagent, and the amount of theophylline in the sample is eye-dent from the data presented in Table VII

Docket No. 2411-A -41-EXAMPLE VIII ~231048 The next series of experiments demonstrates the use of glucose oxidize and glucose and oxygen to generate HOWE from oxygen and the use of cyanide, an inhibitor of peroxides, in an immunoassay for theophylline-sensitized liposomes contain-in peroxides.

event Preparation Theophylline solutions of 2.5, 5, 10, 20, 40, 60 and 100 gel in TRIP buffer were prepared from the stock theophylline solution of Example IV. A 1.0 ml Alcott of each theophylline solution was combined with 1.0 ml of normal rabbit serum Irvine Scientific, Irvine, CA) to obtain theophylline samples of 1.25, 2.5, 5, 10, 20, 30 and 50Jug/ml. Similarly, a 1.0 ml Alcott of TRIP buffer was combined with a 1.0 ml Alcott of normal rabbit serum to prepare a 0Jug/ml theophylline sample.

A solution of 8 x 10 2 M luminol was prepared by dissolve in 719.8 my luminol in about 40 ml of TRIP buffer to which 8 drops of 50% Noah wove) were added. The final volume was adjusted to 50 ml with TRIP buffer. This solution was filtered through a 0.45 u Millipore*filter before use. A stock solution of 4 x 10 2 M luminol was prepared by combining 10.0 ml of 8 x 10 2 M luminol with 10.0 ml of TRIP buffer.
.
A solution of 4.0 mg/ml glucose oxidize (Boehringer Minim, Indianapolis, IN lyophilized grade 1 from Asperqillus nicer) was prepared by dissolving I my ox glucose oxidize in 10.0 ml of TRIP buffer. A 1.0 ml Alcott of this solution was combined with 9.0 ml of TRIP suffer to make a stock solution of 0.4 mg/ml glucose oxidize.

Docket No. 2411-A -42-* Trade Mark ~23~)4L8 A 1/10 dilution of theophylline-sensitized liposomes was made by combining n . 25 ml of fraction 13, Example IV, with 2.25 ml of TRIP buffer.

A reagent containing luminol, glucose oxidize, and the-phylline-sensitized liposomes was prepared by combination of 2~0 ml of the 4 x 10 2 M stock luminol with 2.0 ml of the 1/10 liposome suspension and 4.0 ml of the 0.4 mg/ml glucose oxidize reagent 1).

A solution of 0.2 M glucose was prepared by dissolving 1.82 grams of glucose (Aldrich Milwaukee, WI) in 50.0 ml TRIP
buffer. A stock solution of 4 x 10 2 M glucose, 0.8 my Nan in URIS buffer was prepared by combining 0.5 ml of 0.2 M glucose with 2.0 ml of the 1 my CN-TRIS prepared in Example V.

Anti-theophylline antiserum and guinea pig serum were from the same sources described in Example XV. A 1/20 dilution of anti-theophylline antiserum was prepared by combining 0.25 ml ox rabbit anti-theophylline antiserum with 4.75 ml ox TRIP
buffer. 1/1.25 dilution of complement was prepared by come brining 2.0 ml of Guinea pig serum with 0.5 ml of TRIP buffer A reagent containing glucose, cyanide, anti-theophylline antiserum and complement was prepared by combining 1.0 ml of the 1/20 dilution of rabbit anti-theophylline antiserum with 1.0 ml of the 1/1.25 dilution of guinea pig serum and 2.0 ml or the stock solution of 4 x 10 2 M glucose and 0.8 I Nan in TO
buffer reagent I

essay Procedure _ results A I I Alcott of each of the theophyline samples in 5C~
TV normal rabbit serum was separately combined with 200 I
ox Reagent 1 in a polypropylene tube and mixed. reactions were initiated by addition of 200 I of Reagent 2 to the above arid mixed. Each polypropylene tube was then positioned in a Turner Docket No. 2411-A ~43-I
Model 20 luminometer and the luminescence observed and recorded 15 minutes after initiation of the assay.

Table VIII sets forth the concentration of theophylline in each sample and the luminescence recorded.

TABLE VIII

THEOP~YLLINE
(,u~/ml) LUMINESCENCE
.

lo 25 152 A dose response relationship between the amount of Thea felon in each sample and the recorded luminescence is evil dent from the data presented in Table VIII.

Docket No. 2 411-A -4 4 -

Claims (24)

WHAT IS CLAIMED IS:

1. A composition for determining a ligand in a sample, which composition comprises:
(a) a binding partner for said ligand;
(b) a detection system which has at least two components;
(c) a selectively accessible vesicle having a surface incorporated ligand or ligand analog and a first com-ponent of said detection system therein;
(d) a substance which modifies vesicle accessibility in response to binding of surface-incorporated ligand or ligand analog and the binding partner; and (e) at least one additional component of said detection system which is reactive with said first component to produce a detectable response which is reduced by association of the binding partner and vesicle modi-fying substance with the vesicle.

2. The composition of claim 1 wherein the vesicle exhibits a detectable response in the absence of association of the binding partner and vesicle modifying substance therewith.

3. The composition of claim 2 wherein the detectable response is reduced upon association of the binding partner and vesicle modifying substance with said vesicle.

4. The composition of claim 1 wherein the vesicle is compris-ed of a lipid membrane.

5. The composition of claim 4 wherein the lipid membrane comprises a phospholipid.

6. The composition of claim 4 wherein the lipid membrane incorporates a sterol.

7. The composition of claim 4 wherein the lipid membrane includes an amphiphile to which the ligand or ligand analog is bound.

8. The composition of claim 1 wherein the vesicle is a biological cell membrane.

9. The composition of claim 1 wherein the detection system is luminescent.

10. The composition of claim 1 wherein the first component of the detection system comprises a peroxidatively active sub-stance and the at least one additional component comprises a luminophore.

11. The composition of claim 10 wherein the peroxidatively active substance comprises peroxidase.

12. The composition of claim 10 wherein the at least one addi-tional component further comprises an oxidant for the luminophore, which oxidant is a substrate for the peroxidative-ly active substance.

13. The composition of claim 12 wherein the oxidant is hydrogen peroxide.

14. The composition of claim 10 wherein the at least one additional component further comprises an enzymatic system effective to produce an oxidant for the luminophore.

15. The composition of claim 14 wherein the enzymatic system comprises glucose oxidase and glucose.

16. The composition of claim 1 wherein the detection system further comprises an energy transfer system.

17. The composition of claim 14 wherein the detection system comprises a luminophore and the energy transfer system comprises fluor which absorbs light energy at the wavelength of the luminophore and fluoresces at a different wavelength.

18. The composition of claim 1 which comprises an additional substance which diminishes the luminescence of said detection system upon variation of vesicle accesibility.

19. The composition of claim 18 wherein the additional substance is an inhibitor for the detection system.

20. The composition of claim 19 wherein the inhibitor includes cyanide.

21. The composition of claim 1 wherein the ligand is a hapten or antigen and the binding partner is an antibody therefor.

22. The composition of claim 1 wherein the vesicle modifying substance is complement.

23. A composition for determining a ligand in a sample, which composition comprises:
(a) antibody for said ligand;
(b) complement;
(c) a luminophore;
(d) hydrogen peroxide; and (e) a liposome having a ligand or ligand analog on the surface thereof and peroxidase therein, which liposome exhibits a detectable signal that is reduced upon being bound with said antibody and complement.

24. A method for determining a ligand in a sample, which method comprises combining said sample and the composition of claim 10 or 23 to form a reaction mixture; and observing any reduction of detectable response therein.