CA1237369A - Visualization polymers and their application to diagnostic medicine - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to methods for medicinal diagnosis and to substance for obtaining such diagnoses.
More specifically the substances are visualization polymers of molecules such as proteins, enzymes, and chemically tagged polyols, polyolefins, carbohydrates and natural or synthetic polypeptides, which can be combined with biological material and provide substantial chemical amplification of the quantity of material detected. The specification discloses a method for visualizing the presence of an inorganic or organic target molecule in a biological material, which comprises:
combining said target with a visualization polymer comprising multiple units of a visualization monomer directly bonded together or linked by a coupling agent through chemical groups or backbone moieties of said units;
said visualization monomer having at least one visualization site and being selected from an enzyme, a tagged natural or synthetic polypeptide, a tagged polyol, a tagged polyolefin or a tagged carbohydrate;
said chemical group being a amine group, an oxidized 1,2-diol group, a carboxy group, a mercaptan group, an hydroxy group or a carbon-hydrogen bond;
said backbone moiety being an amide bond, a carbon-carbon bond, a carbon-oxygen bond, or a carbon-hydrogen bond; and said chemical group or backbone moiety being located within said monomer at a position which is at least one atom away from the visualization site of said monomer.

Description

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1, 1, NOVEL VISUALIZATION POLYMERS
li AND THEIR APPLICATION TO DIAGNOSTIC MEDICINE
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2i The invention relates to methods for medicinal 3l~ diagnosis and to ubstances for obtaining such diagnose~.
4 More specifically, the substances are visualization polymer~
of molecules ~uch as proteins, enzymes, and chemically tagged 6If polyols, polyolefins, carbohydrates and natural or synthetic 7I polypeptides, which can be combined with biological material 8 and provide substantial chemical amplification of the 9ll quantity of material detected.
The requirements for a medical diagnostic method, 11 which detects and/or quantifies the presence of biological 12 material, include identification of extremely small 13 quantities and selection of a single species of material from 14l a complex mixture containing ~imilar specieq. In the past, such methods as radiolabeling, radiobioassay and immunoassay 16 techniques have formed the basis for such diagnostic 17 medicine. For example, immunological reagents have been used 18 extensively for detecting and/or quantitating a broad 19 spectrum of molecular species such as proteins, lipids, carbohydrates, steroids, nucleic acids, drugs, carcinogen ~1 antibiotics, inorganic salts etc. Indeed, polyvalent and 22 monoclonal antibodies ars very important diagnostic tools in 23j most areas of clinical medicine today.
24 During the past 40 years, a variety of procedures 25l have been developed to visualize specific antigen-antibody 26 interaction~ fluorimetrically or colorimetrically. Since the 27 utility of immunodiagnostic procedures often depends upon the 28 sensitivity and the specificity with which the target antigen 29 or molecule can be detected, new methods for increasing these detection parameters are highly desirable. The scienti~ic and .~ ~

123'7369 , i ll patent literature is replete with work designed with this 2ll goal in mind. A detailed discussion of the advantage3 and ~1 disadvantages of immunologic methods can be found in any 4j¦ ~tandard textbook on immunocytochemistry; see for example, 51 Lo A. Sternberger, ~Immunohistochemistryn, 2nd Ed.,` John 6 Wiley and Sons, New York, 1979.
7 Immunologic detection methods can utilize direct or 8l indirect vi~ualization technique~ for measurement of the 9j formed immune complex. In general, these methods visually lOj indicate the presence of the complex through use of an entity lljl coupled to the complex which produces a detectable, l~ quantifiable signal such as color, fluorescence, l3l radioactivity, enzymatic action and the like. The more I
14, signal intensity present per complex, the better will be the sensitivity for the presence of a minute quantity of target 16 molecule.

17l Of these methods, the simplest and least sensitive 18l is direct immunofluorescence. In this method, a primary l9~ antibody (or speci~ic ligand-binding protein) is chemically 20i linked to a fluorochrome, such as rhodamine or fluorescein 21 which functions as the signal entity.

22, Indirect immunofluorescence methods, in which a 23~l primary antibody is used unmodified and it9 in turn, i3 241 detected with a fluorescently-labeled secondary antibody, 25l¦ generally will increa~e the detection sensitivity about two 26ll to four-fold over direct methods. An additional thrse to 27!l five fold enhancement in sensitivity has been reported using 28 a "haptene-antibody sandwich" technique~ ~ee Cammi3uli, et 29 al., J Immunol. , 11791695 (1976); Wallace7 et al., J

30~ Immunol Methods, 25, 283 (1979). According to thi3 technique9 .1 1 ! I

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'I , 11l ten to fifteen molecules of a small haptene determinant such 2l¦ a~ 2,4-dinitrophenol are chemically coupled to each primary 311 antibody molecule. Then, by use of a fluorescently-labeled 4,l second antibody which complexes with the haptene molecules~
Sj~ rather than with the primary antibody itself, more of the 6 secondary visualization protein can be bound per antigen 7 site, thus further increasing the sensitivity~
8ll Nakane and associate~; see Nakane, et al., J.
9~ ~ , 22, 1084 (1974), Wilson, et al.
101 "Immunofluorescence and Related Staining Technique3~, W.
11" Knapp, H. Holuban and &. Wick, Eds. Elsevier/North-Holland 12 Biomedical Press, p. 215; have coupled secondary antibodies 13lj to monomeric horseradi3h peroxidaqe and used the catalytic 141 activity of peroxidase enzyme to reveal either the site, or 15ll the amount, of antigen in the test sample. Similar enzymatic 16l assays have been developed with intestinal or bacterial 17 alkaline phosphatase con~ugated secondary antibodies; see 18l Avrameas, Immunochemi~try, 6, 43, (1969); Mason9 i~t. al., J.
19 Clin. Path., 317 454 (1978).
201! The enzymatic signal of this method may occur in 21 either of two ways. Enzymatic conversion of a soluble enzyme 22, substrate into an insoluble, colored product permits the 23` direct localization of the antigen by direct macroscopic 24j visualization or by light microscopic examination.
Alternatively, colorles~ qub~trates can be enzymatically 26~, converted into soluble colored product~ which can be used to 27, quantitate antigen concentration~ by direct colorimetric 2811 analy~is. The latter method iq the ba~is of the Enzyme-29l Linked Immuno-Sorbent Assay (ELISA), which lci widely used in 3~, clinical laboratories around the world; see Sternberger, ~1 ~a.i 3~736~

Il I

l, Immunohi~tochemistry, 2nd Edition, John Wiley and Sons, N.Y.
I

2 I (1979); Engvall, et. al., Immunoche~., 8, 871 (1972);

3 Engvall, et al., J. Immunol~, 109, 129 (1972); Guesdon, et.
41l al~, J. Histochem~ and Cytochem., 27, 1131 (1979); Voller et.
Sj; al., "The Enzyme Linked Immuno Sorbent Assay (ELISA)", S,l Dynatech Laboratories Inc., Alexandria (1979~. 1 7li These enzyme-based detection methods are generally 8i more sensitive than direct or indirect immunofluorescence 9l, methods since the high turnover of substrate by the enzyme lOI continuously accumulates a measurable product over long ll periods of time.
12; To ~urther increase the sensitivity of immunoenzyme l3l assays, Sternberger; see Sternberger, et. al. J. Histochem, I
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14l Cytochem. 18, 315 (1970) developed a three stage peroxidase-antiperoxidase (PAP) assay method. Following the addition o~
16 a primary antibody and a secondary antibody9 which acts as a ., , 17 bridge between the primary antibody and antiperoxidase 18, antibody, a peroxidase-antiperoxidase antibody complex (PAP
9;! complex) is added to the sample prior to the development of i i i the enzymatic reaction. Since the PAP complex contains two 21 immunoglobulins (antiperoxidase antibodies) and three active 22 peroxidase molecules, the net effect i9 to provide more 23 enzyme at the antigen site with which to amplify the 24il detection signal. Although quite u~eful, the PAP detection 25l system ha~ limitations. The secondary "bridge~ antibody mu~t ~6 be used at saturating levelq to en~ure optimal binding of` the 27 PAP complex. Furthermore, the antiperoxida~e and the primary " ~, . i ~" antibody must be of the same, or an immunologically croqs-2~' reacting, species 90 that the secondary antibody will bridge to both. I`
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l~ During the past few years it ha~ been shown that 21¦ the specific and tenaciou~ interaction between biotin, a 3jl small water soluble vitamin, and avidin9 a 687000 dalton 4l glycoprotein from egg white, can be exploited to develop 5,; antigen or ligand detection systems, see Bayer and Wilchek in 6 Voller, et. al.," The Enzyme Linked Immuno Sorbent Assay 7l (ELISA)", Dynatech Laboratories Inc., Alexandria (1979).
8ll Biotin can be covalently coupled to amino, carboxyl, thiol or 9 hydroxyl groups present in proteins, glycoproteins, polysaccharide~, steroids and glycolipids using well lll established chemical reactions; see Guesdon, et. al., J.
12 Histochem. and Cytochem., 27~ 1131 (1979); Sternberger, et.
13, al., J. Histochem. Cytochem., 18, 315 (1970); Bayer9 et. al., 14 Method~ Biochem. Anal., 26, 1, (1980); Bayer, et. al., J. Histochem. Cytochem., 24, 933 (1976); Heitzmann, et. al., 16 Proc. Natl. Acad. Sci. USA, 71, 3537 (1974). Biotin can also -17 be introduced into other macromolecules, such as DNA, RNA and l~ co-enzymes, by enzymatic methods that utilize biotin-labeled 19 nucleotide ~recursors; see Langer, et al., Proc. Natl.
Acad.Sc~. USA, 78, 6633 (1981). Similarly, avidin can be 21 coupled to a host of molecular species by standand chemical 22 reactions; see Sternberger, 1'Immunohistochemistry", 2nd 23 Edition, John Wiley and Sons7 N.Y~ (1979); Nakane, et. al. 7 241 J. Histochem. Cytochem , 22, 1084 (1974); Guesdon, et al , Histochem. and Cytochem , 27, 1131 (1979); Bayer et. al.~
~6 Methods Bioch_m. Anal., 269 1, ~1980). This allows for great 27 flexability in designing detection sy~tems for use in 2B immunology, immunopathology and molecular biology.

29 In 1981 Hsu; ~ee Hsu, et. al., Amer. J. Clin.
30; Path., 75~ 734 (1981); Hsu, et al., J ~ oc~r_ 5/toGbem , _ ~ _ l 29, 577 (1981); reported the use oP avidin-biotinylated 2 ~ horseradish peroxidase complex (ABC) Por antigen detection.
3i In their three-step procedure, the primary antibody

4 incubation is Pollowed by an incubation period with a biotin-labeled secondary antibody and then with the ABC
6 complex, formed by preincubating avidin with a titrated 7 amount of biotinylated peroxidase. Since avidin has four 8 biotin~binding site~ per molecule, at least three peroxidase 9 enzymes can be added to avidin without interfering with its ability to interact with the biotinylated secondary antibody.
ll Hsu and associates; see Hsu et. al., Amer. J. Clin. Path., 12 75, 734 ~1981); Hsu, et. al., J. Histochem. Cytochem., 29, 13 577 (1981); reported that the ABC detection procedure was 4-8 14 times more seasitive in detecting antigens in tissues than either the immunoperoxidase or the PAP detection systems.
16 Madri; see Madri, et. al., Lab. lnvest., 489 98 (1983); has 17 confirmed these observations and shown that the ABC method i~
18 four-Pold more sensitive for antigen detection using an E~ISA
l9 system than either the immunoperoxidase or the PAP
techniques. By all criteria tested, the ABC method is the 21 most sensitive detection procedur0 used in clinical 22 diagnostic labs to date.
23 The limit of sensitivity for the ABC method, 24 however, appears to be 30 to 100 pg of a target molecule ~uch as a protein or nucleic acid. This i~ signiPicantly higher 6 than the upper limit required for detection of a single 27 molecule per cell. Limits for other less sensitive methods 28 are even higher. Accordingly, it i~ an ob~ect of the invention to develop vi~uali~ation methods which substantially improve sensitivity over that provided by known ! !
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1 visualization tschnique~. Yet another object is dsvelopment 2, of a stable, easily manipulated visualization sy~tem which has a long shelf life. Finally, as with any diagno~tic technology, an ultimate goal would be development of a capacity for detecting a single molecule of a species in any 6 given cell.
Summary Of the Invention These and other objects are achieved by the invention which is directed to a method for visualizing the 1~ presence of an inorganic or organic target molecule, a visualization polymer and a detection-visualization complex 12 used in this method and a detection kit for accomplishing 13 practise of the method.
14; According to the invention, the presence of a target molecule may be visualized by combining it with a 16 visualization polymer o~ multiple units of a visualization 17 monomer covalently linked together by polymerization or a 18 couplin~ agent. The target-polymer combination is accomplished through the intermediacy of a detecting agent which is selective for the target molecule and carrie~ the 21 visualization polymer.
22 The visualization polymer provide~ sub~tantial chemical amplification of the quantity of target molecule detected. Each unit of the polymer pos~esseq at least one vi~ualization 3ite and the unit~ are linked in a manner which pre~erve~ the intrinsic activity of the visualization ~ite~

of the unit~. A unit is a vi~ualization monomer which can generate or produce color, fluore~cence, lumine~cence, 2g localization of radioactivity or localizatlon o~ electron dense material. The unitq may be selected from an enzyme or -7- :

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a tagged natural or synthetic polypeptide~ a tagged polyolO a tagged polyolefin, or a tagged carbohydrate.
The units are directly linked by polymerization or indirectly linked by a coupling agent. Direct polymerization or agent coupling bonds chemical groups or monomer backbone moieties of adjacent units. The chemical groupQ or backbone moieties utilized for each unit of polymer will be independently selected from an amine group, an oxidized form of a 1,2-diol group, a carboxy group, a mercaptan group, a 10, hydroxy group or a carbon-hydrogen bond.
Preservation of monomer unit visualization site activity is obtained by forming polymerization bonds or linking chemical groups or backbone moieties of the units which are at least one atom away from the visualization site.
This may be determined by praparing a visualization polymer with each type of coupling agent or with each type of direct ; polymerization process and testing to determine whether 18 additive activity has been produced. Alternatively, if the overall chemical structure of the visualization site can be determined, monomer moieties which do not form part of thi~

structure may be linked by the coupling agent or 2~
polymerization process. For instance, if the site is analyzed and found to contain glycine~ serine and histidine 24 l I but not lysine or a sugar, lysine or a sugar may be used as 25 i the chemical group for linking.
26 !

27 The visualization sites of the monomerQ can be site of biological activity. For example, 3ites for enzymatlc action will provid~ visualization when reacted with an appropriate substrate. In this manner, the visualization sites can be utilized to generate soluble or in oluble bodies ~3~73~9 ~.
, .

l of color, fluorescence, luminei3cence, radioactivity or high 2jl 1 electron densitv -hich can be measured and correlated with 1, the quantity of target moleculei3 detected~

The sites may also be created chemically.

Combining a natural or synthetic polypeptide, polyol, polyolefin or carbohydrate with a visualization tag selected from a fluorescent chemical group, a dye, a radioactive group, a photon emitter (a luminescent group) or an electron g :
dense moiety will produce monomer units which can be 10' visualized. The tag may be present at an equivalent ratio 11 !
relative to each unit. It is preferred, however, to have a multiple number of tags per unit.

The unlts of the visualization polymer are joined 14 j ~ together by direct polymerization procesi3 bonding or by coupling agent linking. Direct polymerization produces interbonding o~ available chemical groups or backbone moieties in adjacent units. For example, oxidative enzymeisi i such as horseradish peroxidase can be used to polymerize monomer units by oxidative cross-linking.

Alternatively, a coupling agent, derived from a 21~
bifunctional or multifunctional organic cross-linking reagent, bond~ with the appropriate chemical group or backbone moiety of the units~ In this context the term `I ncoupling agent~ denotei~ the linkage group after ~onding and 25 i the term cross-linking reagent denotes the linkage eompound 26 i before bonding.

The cro~s linklng reagent has generic formula I:

i A B
29 ~ \ R1 /
30, - E
~9--i ~ ;~3~36~

Reactive group~ A, B and E of formula I are independently ~' qelected from hydrogen, a carboxylic acid group, an acid jl halide, an activated ester, a mixed anhydride, an acyl imidazole, an N-(carbonyloxy)imide group, an iminoester, a primary amine, an aldehyde group, an alphahalomethylcarbonyl group, a hydrazine group, an acyl hydrazide group, an azide group or an N-maleimide group. At least two of A, B and E
are other than hydrogen. Multifunctional cro~s-linking 9 ' reagentst with more than three reactive groups which are qimilar to A, B and E, are al o within the scope of the invention. These additional reactive group~ will be independently selected from the foregoing definitions of A, B
and E
14 R1 of formula I is an aliphatic group of at lea~t two carbon3 or an aromatic group of at least six carbon~.

Choice of the reactive group~ of formula I will ; depend upon the selection of the chemical groups or baokbone moietieq of the monomer units which are to be linked. Each kind of chemical group or backbone moiety will react with it~
corresponding appropriate reactive group or groups of formula I. An amine group will react with a carboxylic acid group, an acid haIide, an activated e~ter, a mixed anhydride, an , acyl imidazole, an N-(carbonyloxy)imide group, an iminoester9 24 l i an azide or an aldehyde group. An oxidized 1, 2-diol group ~a dialdehyde) will react with a primary amine, a hydrazine 26 ' groupy an azide or an acyl hydrazide group. A carboxy group ;~ will react with a primary amine, a hydrazine group, an azide or an acyl hydrazide groupO A mercaptan group will react with an alphahalomethylcarbonyl group or an N-maleimide group. An hydroxy group will react with a carboxylic acid i ~373 group, an acid halide, an activated ester, a mixed anhydride, I an acyl imidazole or an N-(carbonyloxy)imide. A carbon-l hydrogen bond will react with an azide (nitrene)~
4 l .
Accordlng to the method of the invention, there is utilized a detecting agent which is specific for the target molecules. The agent directly or indirectly carries the visualization polymer to the target. The detecting agent may be an antibody, a lectin, a DNA repressor protein9 a stereospecific receptor-protein, a high affinity enzyme; a 10 ' sequence specific polynucleotide binding protein, avidin, streptavidin, a hormone or a complementary polynucleotide sequence.
13 The detecting agent may carry the visualization polymer by direct covalent bonding with the polymer, or indirect bonding through an intermediate covalent bonding group. The detecting agent may also carry the polymer through an intermediate ligand binding complex which arrangement may be direct or indirect. In the direct arrangement, the agent acts as a ligand binding compound also and the corresponding ligand is covalently bonded to the i visualization polymer. In the indirect arrangement, a fir~t ligand is bound to the agent, a second ligand is bound to the I polymer and they are sandwiched with a ligand binding 24 !
Il compound ~uch that the first and second ligands ~unction as br~dges complexing with the compound.

Ligand binding compoundq include an antibody, lectin, avidin, streptavidin, a high aPfinity enzyme, a ~8 sequence specific polynucleotide binding protein or a 23 ;l complementary polynucleotide sequence. Ligand~ will be the appropriate ones forming complexes with each type of ~l~3~
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1 compound. They include antigen, specific sugar, biotin, I' iminobiotin, qpeci~ic ~ubstrate, polynucleotide and I complementary polynucleotide respectively.
4 ~
A preferred detecting agent-visualization polymer carrying arrangement according to the invention i5 the indirect arrangement wherein the detecting agent is an antibody, a complementary polynucleotide sequence or is a lectin; the ligand binding compound is avidin or 9 ' ~treptavidin, and the first and second ligands are independently selected from biotin or iminobiotin, N-(omega alkanoyl) amido ~biotin or iminobiotin] wherein the alkanoyl ~ group i~ 4 to 20 carbons in length or and N-(omega-oligomer) 13 amido [biotin or iminobiotin] wherein the oligomer is a polyol, polyamide or polyvinyl group of 2 to 30 unit~ in length. A preferred ratio of vi~ualization polymer to avidin 16 or strepavidin in any of these ligand complexes i9 about 3 to In this preferred indirect arrangement, when the detecting agent i~ a lectin, the target molecule will be a corresponding appropriate sugar. When it is an antibody, the 21 target molecule will be an antigen or haptene which complexes with that antibody. When it is a polynucleotide sequence, the target will be a complementary polynucleotide sequence which hybridizes with the agent.
25 Ij l A preferred method according to the invention utilize~ the foregoing preferred lndirect arrangement An e~pecially preferred method for detection of target moleculeQ i3 built upon the foregoing preferred arrangement; however, it employ3 a second bridging component.
30 !
The complex, i.e., avidin or streptavidin~(biotin ligand)-i --t2--1 ~23,~369 1 visualization polymer, i~ used to complex with ~ biotin labelled second antibody. The second antibody is a general il reagent for the firqt antibody detecting agent which in turn 4~i .
is specific for the target. The first antibody i~ incubated with the target to form an antigen-antibody conjugate. Then the second antibody is incubated with this conjugate.
Following the second incubation, the visualization polymer ` complex is added which binds to the second antibody and g provides visualization.
1.
Yet another preferred method, according to the 11 invention, also utilizes the preferred indirect complexing 12~
ligand arrangement. In this arrangement, the detecting agert is a complementary polynucleotide sequence and the target is the corresponding native polynucleotide sequence which will ~ hybridize with the complementary sequence. The detecting agent and the visualization polymer are labeled with a biotin I or iminobiotin group. A complex of avidin or streptavidin-18(biotin ligand)-visualization polymer is formed The labelled polynucleotide detecting agent is added to the complex biological mixture containing the native polynucleotide sequence to be detected. Hybridization is allowed to take place, then the complex is added which bind~ i I to the hybridized and labelled polynucleotide detecting agent li and which provides visualization~

I Especially preferred carrying arrangements include 26 l ;! those of the two foregoing preferred methods.

The complexes between the ligand carrying compound and the visualization polymer bonded to the corresponding 29, ligand are al~o included within the invention. These 30i complexes are described above a~ part of the carrying :

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arrangement of the invention.
2 l I Preferred detection methods and preferred visualization polymers include polymers having multiple units of an enzyme or multiple units of a natural or synthetic S
polypeptide or polyolefin chemically bonded to a tag selected from a ~luorescent group, a dye, a luminescent group or an electron dense group. Preferred enzymes include alkaline phosphatase, peroxidase, galactosidase, glucose oxidase, acid g phosphatase and luciferase. Preferred polypeptides include polyamides of dicarboxylic acids and diamine, polyamides, 11 ' oligomers and copolymers of alpha amino acids such as glycine, lysine, aspartic acid, cysteine, ornithine and the l~ like. Polyolefins include polyacylamide 9 polyacrylic acid, polymaleic acid, poly(hydroxyethylacrylic ester) and the like. These polypeptides and polyolefins will be tagged with such groups as fluorescein, rhodamine, a diazo dye, colloidal gold, luciferin, radioactive iodine and the like.

For the preparation of the visualization polymer, preferred coupling agent-chemical group bonding arrangements ~ include:

1~ A diacyl or di(iminoester) derivative of an aliphatic dicarboxylic acid of from 4 to 20 carbons which j will form amide or amidine bonds with epsilan or primary 2~ l , amine groups of monomer~ functioning a~ unit~ of the polymar.
25 , i 2. A reactive diaoyl or dihydrazide derivative of ~6l~
an aliphatic dicarboxylic acid o~ from 4 to 20 carbon~ or an aliphatic dihydrazine o~ from 4 to 20 carbon~ which will form amide or hydrazone groups with 1,2-diol groups of monomer~
29 ;
functioning as.units of the polymer when the 1,2-diol is 3~
oxidized to a dialdehyde, or which will for~ amide groups ' !

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1 with carboxylic acid groups of monomer~ functioning a~ unit3 I of the polymer.
3 1l 3. A reactive olefin derivative of an N-alkyl bis(maleimide) of from 4 to 20 carbons in the alkyl group which will form diqulfide groups with mercaptan group~
` present in monomer~ functioning as units of the polymer.
4. A reactive aliphatic heterobifunctional reagent substituted with an N-maleimide group and either an iminoester or an N-(carbonyloxy)imide group wherein the aliphatic chain length is from 4 to 20 carbons, which will form a ~ulfide group with a mercaptan group oP a monomer 12 functioning as a unit of the polymer and will form an amidine or amide group with an amine group an adjacent monomer functioning as a unit of the polymer.

5. A reactive aliphatic heterobifunctional reagent substituted with Schiff base protected amine group and an acyl or iminoester derivative group of a carboxvlic acid wherein the aliphatic chain length is from 4 to 20 carbon~, which will form an amide or amidine bond with an amine group of a monomer functioning a~ a unit of the polymer, and after 21 removal of the Schiff base protecting group, will form an amide bond by carbonyl dimida~ole or diimide coupling with a carboxyl group of an adjacent monomer functioning a3 a unit of the polymer.
, 6. A trifunctional lysyl ly~ine reagent which will 2~ ;
form imine or amide bond~ with oxidi~ed 1,2-diol groups or carboxylic acid groupY respectively which are present in monomers functioning a~ unit~ of polymer.
29 ;
The invention i~ a~ well directed to a detection kit for analysis of the preqence of a target molecule , i , 1l especially in a biological material. The kit includeq , metered quantities of a mixture of a detection-vi~ualization ;I complex or the components thereof as set ~orth above 9 which j is specific for the target molecule, and a standardized quantity of the same visualization polymer. The kit may be in the form of a colorimetric, fluorescent, luminescent or radioactive indicator in which the test quantities and standards are in aqueous solution and in appropriate containers for colorimetric, fluorescent, luminescent or 10 ' i radioactive analysis. The kit may also be in the form of 11 test papers and standard papers such as nitrocellulose which will permit vi~ualization of the target molecules.
13 Brief Description Of The Figures . , , The Figures illustrate the results of detection tests conducted according to the invention.
16 Figure 1 shows detection of Biotin labelled DNA on ~7 nitrocellulose dot blots using a polyenzyme/avidin complex.

Figure 2 shows a comparison study using biotin free 19 "
DNA probe~.
Figure 3 shows detection of Biotin labelled DNA by the Southern Blot Method using a polyenzyme/avidin complex.

Figure 4 shows complementary hybridization detection of human alpha and beta globin geneY by the , Southern Blot Method usine a colorimetric visualization and ; vi~ualizat~on polymer according to the invention.

Figure 5 ~hows comparative detection results produced by vi~ualizing a biotinylated protein with an enzyme (A) and a polyenzyme (B) according to the invention.
29, Figure 6 ~hows the results of a PAP detection technique for detection o~ protein.
~16 ;

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2~ Figure 7 show~ the results of the poly ABAP
1 technique for detection of protein according to the 3li invention.

Detailed Description Of The Invention The visualization polymers and complexes of the present invention detect and chemically amplify the presence of minute quantities of inorganic or organic target molecules which may be found in biological material. Generally, the detection is based upon interaction between the polymer, its complex and the target molecule to be detected. The polymer 11 is carried in a complex carrying arrangement which can bind 12 with ~pecific target molecules and exclude others.

Quantitative determination of the target is ~ade by measuring 14 the amount of polymer pre~ent in the association formed between the target molecule and carrying arrangement. Signal amplification i~ provided by the multiple units of the polymer in each association.
18 The monomer units of the polymer are an important feature providing visualization of the target carrying arrangement association. The units can contain visualization 21 tags or can react with a qubstrate which can be utilized a~ a means for quantitative measurement. This measurement may be accomplished by production of a readily identi~iable ~ubstrate i product or production of a ~pectroscopic ~ignal, as well ~9 other, similar types of nondestructive quantitative analytic method~ for measurement. Pre~erably, the visualization will be based upon the production of oolor~ ~luorescence, luminescence, radioactivity7 high electron density as well as 2~ ;
other forms of spectroscopic measurements.
When the units are enzymes they can generate i -17;

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I , l productq which are capable of producing such spectroscopic 2!1 :
mea3urement. For example, they may catalyze reaction of sub~trates to produce colored, fluorescent, luminescent, electron dense or radioactive products.
Alternatively, the tagged units may be directly utilized as tools for spectroscopic measurement. For example 9 the natural or synthetic polypeptides, polyols, polyole~ins or carbohydrates may be tagged with chemical groups which have 9' coloration, fluorescent, luminescent, electron dense or 10: `
radioactive propertiesO These may then be used for spectroscopic measurement.

Enzymes and tagged polypeptides, polyolsl polyolefins or carbohydrates possessing the foregoing properties are well-known as means for spectroscopic quantification. When placed in an an appropriate spectrometer, the enzymatic ~substrate or tag will cause a spectrographic change which will indicate the quantity of target present. This process is commonly referred to as visualization and the 3pectral change is termed tha signal ~0 produced by the visualization group (the substrate or tag).

The quantity of target to be detected usually will be minute and if the signal from the complex-target as~ociation were produced on an equivalent basis 7 it al~o 24 , ~I would be extremely weak. However7 the carrying arrangement 25 !
and its vi~ualization polymer~ chemically amplify the signal 26 i so that minute quantities of target will produce a strong, 27' , readily determined signal. Ampli~ication is achieved by the polymer becau~e it comprise~ multiple units of the visualization monomer. The signal provided by each unit i~
maintained by the polymer. Consequently, its signal i3 the i 1' _18- !

3~

! sum of the signals of its units. In addition, the carrying arrangement may contain multiple number~ of polymer Although it is not necessary~ this multiple arrangement is preferred ~ince it provideq further amplification.
The visualization polymer of the invention compri~es multiple units of monomer directly bonded together or indirectly linked together by a coupling agent bonded to chemical groups or backbone moieties of the units. Each unit also possesses a site or sites which provide the visualization signal. That is it may be a site for enzymatic action or a site to which a visualization tag or tags are attached. The visualization signal activity of the polymer depends upon production of a signal by each monomer unit. Accordinglyt the visualization site or sites should be substantially preserved in its or their original Por~ so that the site activity is not 16 substantially decrea~ed. It follows that chemical modification 17 of the monomer units should be conducted in a manner which does not substantially affect the site or sites.
19 To this end, the direct bonding or coupling agent linkage should ~oin chemical groups or backbone moieties of the unit~ which are at least one atom and preferrably at least 3 to 5 atoms away from the viqualization site or ~ite~. Also, the choice of chemical groups or backbone moieties for direct bonding or linking with coupling agent ~hould be limited to those which are not present within the sita or which are not necessary for ~ite conformation and three dimensional configuration. This choice will be more important for enzyme protein~ than for tagged natural or synthetlc polypeptide~
29i ~ polyols, polyolefin~ or carbohydrates; however, interference with the production of tag ~luore~cence, luminescence9 _19_ 3~3 ' '.
.

coloration, radioactivity or high electron density should also be avoided.
Generally, these site preservation requirements may be met in several wayq. If the types of biochemical substructures or chemical re~idues making up the monomer structure are known, then one which is not part of the visualization site may be chosen as the structure containing the reactive chemical groups or backbone moieties for coupling. Usually, however, a ~emi-emperic method will be used for choice o~ the appropriate reactive chemical groups or 11 backbone moieties-According to the substructure/residue method, the chemical construction of the monomer units will be investigated. The monomer backbone substituted groups and functional structures such as sugar groups, lipids, oligomer side chains and the like which are not necessary for vi~ualization site action will be identified. Typically, this would be determined by removal modification or modification of such substructures and study o~ the activity of the resulting ~ product. Chemical groups or backbone moieties present primarily within these substructures may then be used for direct bonding or indirect linking with the coupling agent.

`I For example, the ugar group~ of a glycoprotein which are not 2~
!I necessary for enzymatic activity can be oxidized to dialdehyde 25 l groups and reacted with a hydrazine coupling agent to form the 2~ ~
visualization polymer.

I~ the chemical ~equence oP the monomer, such as the amino acid sequence of a protein, can be determined, this may also be utili~ed to guide direct bonding or indirect linking.

Analy3is o~ the sequence for the active ~ite as well as the .~ .
--2 0_ ., ., three di~ensional configuration will show which monomer structural subunit3 are not es~ential to functioning of the site and/or not present within it. The reactive chemical groups or backbone moieties of these ~ubunits may be used for bonding or linking with the coupling agent. For example, if

6 i I the monomer is a protein and it is found to contain a , dipeptide side chain ending with cysteine, the mercaptan group of the cysteine may be cross-linked to oysteine of another similar protein by reaction with bi3 (N-butylenylmaleimide).
According to the semi-emperic method, the reactive chemical group3 and backbone moieties of the monomer can be determined by appropriate spectrographic and chemical analysi~. These include technique~ such a3 NMR, IR, chemic~l 1~ :
derivatization, electrophoresis, osmometry ? amino acid analysis, elemental analysi3, ma3~ spectrometry and the like.

The groups and moieties identified may include amine groups, 17;
mercaptan groups, carboxyl groups, hydroxyl groups, sugar groups, carbohydrate groups, ester groups, lipid groups, and amide bonds, labile carbon-carbon bond~ and carbon hydrogen bonds the like. Other measurement~ such as the relation of derivatization and site activity, relation o~ pH and ~ite ; activity and type oP site reaction produced in the ca~e of an !l enzyme will help determine a priority ~or the Punctional 24 !1 ,I group~ ba~ed upon the probability of their presence withln the 25 j vicinity of the active ~ite. A typical priority will be 1. an epsilon or primary amine group, 2. sugar group, 3~ carboxyl group, 4. mercaptan group~ 5. hydroxyl group, 6. lipid group~
28 !
i If derivatization of amine groups such a~ tho~e of ly~ine re~idues produce~ a derivatized product devoid of ~lte 30l, activ~ty, then the foregoing priority will change and the !~
!

1~:3~3~

, 1 amine group will be last.

Under usual emperic procedures, qeveral ver~ionq of 3 l polymer will be prepared u~ing a ~electlon of several of the reactive chemical group~ or baokbone moietie~. The activities of the several versions are then tested and the one selected of which has the highest activity. Typically, the selection of chemical groups or backbone moieties will encompass three or four types which are least likely to affect the activity of 9 '.
~ the visualization site. Each type of reactive chemical group 10 - j ; or backone moiety may eventually be tried if resultq with the first few are unsatisfactory. Emperic examination of each Yersion of polymer will allow identification of the one with the highest activity.

The monomer unit~ having vi~ualization sites which are very ~enqitive to the chemical group/backbone moiety bonding arrangement are enzymes. The catalytic site typically will have a conformation closely fitting the substrate and chemical modification which disturbs the three dimensional 19 ~ .
configuration of the catalytic ~ite may adversely affect the activity of the polymer. Following the Poregoing procedures, enzyme site activity can be preserved. ~Furthermore, the enzyme catalytic site may be protected during bonding or ; linking by reversably binding it with 3ubstrate.
24 !
;I Tagged natural or synthetic polypeptide 7 polyol 9 25 i polyolefin or carbohydrate will have visualization sites ~hioh are ~ubqtantially le~ sensitive to the chemical group/backbone moiety bonding arrangement. The fluoreqcent ! group, dye, luminescent group7 radioactive group or electron 29, den~e group which acts as the tag typically will not be sub~ect to variation~ in activity when ad~acent chemical ~3~3 .

.
groups or backbone moieties are directly bonded or indirectly 2,i linked with coupling agent. Care must be taken, however, to 3 l assure that the direct bonding or coupling agent does not also ! react or otherwise interfere with the tag to lessen its visualization activity. Moreover, if the tag is to be converted to an active group after the visualization polymer-target association is made9 then the position of the chemical group or backbone moiety linkage should not inter~ere with the conversion.
Generally, the chemical groups and backbone moieties should be at least one atom away from the visualization site.

Enzyme protein monomer~ will be bonded or linked preferably about 3 to 5 atoms away from the visualization site.

The visualization polymer structure is multiple units of monomer either directly interbonded or cross-linked by coupling agent. The structural and ~unctional character of the poly~er will be ~imilar to that of the monomer units. The number Or units per polymer will depend upon the extent of ~ coupling~ the stability of the resulting polymer~ the reactivity o~ the chemical groups or backbone moieties relative to the polymer chain length and the position of the groups or moieties along the monomer backbone.

Generally, the number of units inoorporated into the polymer may vary from as few a~ two to three to as many a~
~l i ~l thirty to one hundred per polymer. Higher multiples are al~o po~ible provided that the polymer chain length is not of an order which will render the polymer extremely insoluble in aqueous ~olution or will be extremely suseptible toward ! mechanical cleavage.

; The polymer may be linear or it may be branched.

-23_ .

There may be single or multiple coupling between two adacent units. Coupling may occur at any point along the unit chain so that adjacent unit~ may lie end to end, or may partially or fully overlap. A~ a result, the three dimensional ~tructure of the polymer may have all of these features. It may be linear but more typically will be a combination oY linear and branching unit~. Partial overlap will typically occur and multiple coupling will al~o be present.
The accessibility of the chemical groups or backbone moieties will also affect polymer length. If they are buried within the monomer structure, steric inhibition will tend to hinder coupling of a high number of monomer units. This 13 effect may be compensated by use of coupling agents having a chain length greater than about ten carbons in length. In a more usual arrangement, the reactive chemical groups or backbone moieties will be readily accessible and will lie either within the proximate ends of the monomer chain or will 18 lie within an accessible side chain or group which extends away from the inner region of the three dimensional structure ` of the monomer. Thls type of group or moie~y will be amenable toward coupling of high multiples of monomer units.` Typically, direct bonding or any chain length of coupling agent may be ' employed. However, coupling readily accessible groups or 24 j moietie~ with agents which will hold apart the unit~ o~ the I polymer will sometimes provide advantageO This will allow 26;
facile approach of sub~trate or reactant and will prevent adverse interaction among the units of the polymer.
28~
Typically, agents having a carbon chain length of from about 4 ~ to about 20 carbons will be pre~erred.
The coupling agent linking monomer units togsther i i .~ i ~:3~3~i9 , 1 i ; generally iis derived from a bifunctional or multifunctional , . l !1 organic cross-linking reagent of foregoing formula I. In thiis 3!, context, the term coupling agent will indicate the group ln its coupled form with a chemical group or backbone moiety.
The term cross-linking reagent will indicate the chemical form of the agent before it i~i reacted with a chemical group or backbone moiety.
81' i ,; Choice of the coupling agent/croqs-linking reagent I wlll depend upon the choice of the reactive chemical group or 10 i' backbone moiety to be coupled and the agent chain length which will avoid intraunit interference within the polymer. Scheme I illui~trates the general plan for reagent and chemical group or backbone moiety reaction.
1~ ., In each of reactions 1-6 of Scheme 1, the bi or multifunctional cross-linking reagent of formula I is used.
The generic formula for this reagent is 18 R1 / B f E

wherein A, B and E are reactive groups as specified above and R is a~ specified above. In the reactioniq, a specific reactive group for A and its reaction with a monomer chemical 23`
,j group or backbone moiety are given. Groupi~ B and E, which 24~1 ,' are not designated, may be the same, may be chosen Prom any 25 j l of the other reactive groups, or one of B and E may be 26 l ' hydrogen. In this manner, the reaction~ illui~trate croisis 27' j linking reactions with homo- (bl or multi) funotional reagents wherein the reactive groupi3 are the same and with 2ig ~, hetero- (bi or multi) Punctional reagents wherein the reactive group~ are different~
-25_ ~3~36~ 1 R1 O~ formula I u~ually will be ~ubi3tantially linear and will have little, if any, branching. Preferrably, Rl i3 an alkylenyl group of the formula -(CH2)~-; a cycloalkylenyl group of the formula -CH[(CH2)k]2 CH-; or an aryl group of the 6 2)1 C6H4-(CH2)m- wherein j is an integer of fro about 2 to about 30, k i9 an integer from about 0 to 6 and the ~um of k'~ will be 4,5,6,7 or 89 and 1 and m are integers independently selected ~rom 0 to about 20. Ei~pecially preferred R1 groupi3 will include alkylenyl having j from 3 to 14 and aryl having 1 and m from 0 to 5 and having a para sub~titution. Preferred embodimenti3 include propylenyl 9 butylenyl, hexylenyl, nonylenyl undecylenyl, dodecylenyl cyclohexylenyl, phenylenyl or xylenyl.
14 Reactioni3 1A, 1B, and 1C of Scheme I show the coupling of a free amine chemical group. Usually, thii~ will be an epsilon or primary amine group of an alpha amino acid re~idue o~ the protein unit. Example~ of amino acid~

containing an ep3ilon amino gFOUpS include lysine, and arginine. Examples of amino acid~ with primary amine groups include the twenty alpha amino acids typically found in protein. Amine groups on other kindi3 of monomer~ such as amino ~u~ars or aminonucleotides can also be coupled with this ,I reaction.
24 I The reagent~ of formula IlA, which are used to 25' l perform reaction 1A, include those known carboxylic acid derivative3 which will react wlth amines to produce amides~

Included are acyl halide3, mixed anhydrideis, activated e~ter~, i~ acyl imidazole~ derived from carbonyldimidazole, N-~ oxa~uccinimides produced by the dehydratlon reaction of the 30 i j correi3ponding carboxylic acid and an N-hydroxy~uccinimide with ; -26-!

~L~3~736 l a diimide dehydration reagent, amide formation with j dehydrating reagents such as diimides, phosphorus pentoxide in organic 30lvent, and pho~phorus oxychloride. These group3 are indicated as X in reaction 1A .
Generally, the~e acid derivatives may be prepared by condensation of the corresponding carboxylic acid and X group reagent. They may be reacted with the amine chemical group according to reaction lA, u~ing aqueous or polar organic ~ solvent under mild conditions. The method~ and u3e of these reagent~ are known; see for example "Reagents For Organic Synthesis", L. Fiezer, M. Fiezer, Yol. 1-8, Wiley ~ Son;

"Cros~ Linking Reagents" (1980 Ed.), Pierce Biochemical Reagent Catalog, Pierce Chemical Co., Rockford Ill. and ; 14 references therein 9 or "Advanced Organic Chemistry~ J. March, lS
McGraw Hill (1968).

Reaction 1B ~how~ coupling of an iminoester salt reagent of formula IlB with an amine chemical group to produce 1l an amidine coupling agent linkage. This method i3 al~o known in the art. The reagent may be generated from the acidic ; alcoholy~is of the corresponding nitrile. The amidine formation reaction may be conducted in aqueous or polar organic ~olvent under mild conditions. The methods and procedures are known; see for example Lockhart, et. al.~ Can 24 ~
i J. Biochem., 53, 861-867 (1975) and Pierce Biochemical Rea~ent 25 l l Catalog and reference~ therein, ~upra.
26 !
Reaction lC shows condensation of an amine chemical group with an aldehyde reagent of formula I1C to form a bi~
28 ;
I Schiff ba~e (imine). Examples include glutaraldehyde and other 29l ti~ue fixing reagents. Conditions include u~e of polar 30 j organic solvent and mild temperatures. These methods are !
. --27 i ~I Z,37~
., ' . .

l I known in the art.
2;1 j Reactions 2A, 2B and 2C of Scheme I show the 3 i Il conden~ation coupling of an aldehyde chemical group with 4 !
amine~ and amine derivative reagents to form imine and imine derivative compounds. Theqe reagents and reactions include primary amine reagents of formula I2A which react to form a Schiff base (imine), see reaction 2A; ~ubstituted hydrazine reagents of formula I2B which react to form substituted ! hydrazone5~ see reaction 2B; and acyl hydrazide reagents of formula I2C which react to form acyl hydrazones, see reaction 11 ' 2C. The aldehyde functional moiety will be generated 9 in turn~ by oxidative cleavage of any 1,2-diol (glycol) group within the monomer unit except that oxidative cleavage of glycol groups which would re~ult in cleavage of the monomer unit chain cannot be u~ed. Usually, the glycol group will be found on a side chain attached to the monomer backbone.
17' , Example~ include carbohydrate and ~tarch groups as well as dihydroxy alkyl, cycloalkyl and acylalkyl groups.

` Conditions for reaction 2A, 2B and 2C include u~e o~

aqueous or polar organic solvent3 and mild to moderate temperatures. These method~ and procedures are well known in the art, see for example 'IBasic Principles Of Organic Chemistry", Roberts and Ca~erio, W A Benjamin t1965) or i "Qualitative Organic Analy~is," Shriner and Fu~on, Wiley Int erqc i ence ( 19 6 6 ) o 26 ll Reactions 3A and 3B of Scheme I show the coupling of a mercaptan ~hemical group with cross-linking reagent I3A and I3B to form the polymer lin~ed by ~ulfide groups. Reaction 3A

i is condensation of the mercaptan chemical group with alpha 30, halomethylcarbonyl reagent I3A wherein Y is a halogen to form ~28-~Z3~9 ;

carbonyl methylene sulfide P3A; see, for example, Hixon7 et al., Biochemistry, 14, 425 (1975). Reaction 3B is coupling of the mercaptan chemical group with maleimide reagent I3B to form succinyl 3-~ulfide P3P.
The mercaptan groups will be found within the monomer unit as part of a cysteine, glutathione, cystine or similar amino acid unit. They may also be present as a derivative of a sugar or lipid group.

Conditions for reaction 3A will include aqueous or .
polar organic solvent 9 an acid scavanger such as pyridine and 11 ' `
mild to moderate temperatures. Conditions for reaction 3B

will include aqueous or polar organic solvent, optional mild acid catalyst and mild to moderate temperatures. These methods and procedures are known in the art, see Pierce Biochemical catalog and references therein supra.

Reactions 4A and B show the condensation coupling o~

a carboxylic acid chenlical group and an amine or acylhydrazide reagent. Reagent I4A may be coupled with the carboxylic acid group directly through the use of compounds such as diimide or carbonyl diimidazole, see the foregoing des¢ription for reaction lA. It may also be coupled by forming a carboxylic acid derivative such as the mixed anhydride, an activated - !; ester or an acyl halide. Reagent I4B may be coupled with the 24 l oarboxylic acid group through use of a dehydrating reagent I such aQ a carbodiimide; see reaction lA. Carboxylic acid 26~
containing substituents of monomer units would provide the carboxylic acid group directly or may be oxidized to provide it. Theae include amino acid residues such as aspartic acid, 2~
; glutamic acid as well as oxidized forms of sugar side chains.
Lipids, carbohydrates and olefinic carboxylic acidq are also _~9_ .
. .

! . I
~3~3~

included. These methods are known in the art, see for example nOrganic Syntheses", Wiley, New York, Coll. Vol l-V.
Reaction 5 shows esterification of an hydroxy function moiety with an activated carboxylic acid reagent I5.
These would include the same reagents as given for formula I1A. In this synthetic method, if there are free, reactive amine groups of the monomer unit, they could ~irst be protected with a removable protecting group such as a Schiff base, i.e., condensation of the amine groups with an aromatic aldehyde such as p-methoxybenzaldehyde or benzaldehyde which can be removed with dilute hydrogen chloride in acetone.
12 Other known amine protecting groups may alQo be used. These 13 include dinitrofluorobenzene, t-butoxy groups and ! organosilanes.
After protection, esterfication is conducted using 16 the activated acid reagent. Monomer unit residues which will 17 esterify in thi~ fashion will include amino acid residues of serine, threonine 9 hydroxylysine, tyrosine, thyroxine 9 hydroxyproline, and the likeO Other residues include 21 carbohydrate, starch, lipid and olefinic residues with hydroxyl substitution~O These include hexo~es, pen`tose3, ~ dextrans, amyloses, glycerol~, fatty acid derivatives, methylhydroxymethacrylate, hydroxymethyl Acrylate and similar 24 l Compounds.
Conditions for the esterfication include aqueou~ or polar organic solvent~ and mild to moderate temperature~.

These method~ and procedure~ are known in the art; see, for example, the foregoing treati~es on organLc ~ynthesi~.
Reaction 6 show~ insertion of a nitrene from azide reagent I6 into a carbon hydrogen bond. This reaction is i -30-, .

~L~3~3~i~

' :

nonspecific and can take place at any point within the unit.
Since the nitrene i3 ~hort lived, readily accessible carbon hydrogen bond~ will be attacked first. Thi~ reaction i9 e~pecially useful for coupling of the tagged natural or synthetic polypeptides polyols, polyolefin~ and carbohydrates as well as for enzymes having C-H bonds which are readily accessible and not involved with site activity. I
The condition~ for reaction include polar organic solvent and irradiation with 300-400 nm light. The methods and procedure~ for this reaction are known, see for example Bisson, J. Biol. Chem., 253 1874-1880 (1978) or Pierce Biochemical Catalog and references therein, supra.
13 Reaction 7 is the direct bonding polymerization of the monomer units. It may be accompliqhed by enzymtic oxidative cross-linking, photolytic free radical generation and cross-linking or free radical initiation with such reagent~ as persulfite, hydrogen peroxide and triplet oxygen.

Table 1 lists the types of group~ which may function as X, Y, and X' in formulas I1A, I3A and I5 of Scheme 1. These have been described in conjunction with the explanation of each reaction.

25 j 26!!

ll -31 1 ' i, ~

'736~

, .
.

1.,. Scheme I
2 Bonding Or Linking Reaction: I
3ll1 lA. RNH2 ' XOC-R1~BE) ~ RNHCO-Rl~BE) i, 4 IlA PlA

6 lB. RNH2 ~ H2N-C(OMe)-R1(BE) ~ RNHC~=NH)-R1(BE)

7,l IlB PlE
9, lC. RNH2 + OHC-R1(BE) > RN-CH-R1(BE) 10 IlC PlC

12 2A. RCHO = H2N-Rl (BEj RCH=N-Rl(BE) 13. I2B

15 2B. RCHO + H2N-NH-R1(BE) > RCH-NNH-Rl(BE) f 18 2C~ RCHO ~ H2NNHCO-Rl(BE~ ~ RCH=NNHCO-Rl(BE) 21 3A. RSH ~ YCH2CO-R1(B~) RS-CH2CO-Rl(BE) 22 o~ 1 23 33. ~SH ~ C N-R1(BE) ~ RS ~ N-R (EE) 25i I3B P3B
26 4A. RC02H + H2N Rl(BE) ~ RCONH-R1(BE) 27l I4A P4A
~8'1 29, 4B. RC02H + H~NNHCO-Rl(BE) ~ RCONHNHCORl(BE) il 'i 'I 32-37~6~3 f ~I .
1l Scheme I (continued) 2 5. ROH + X'OC-R1(BE) ROOC-R1(BE) 5~ 6. RC-H + N3-R1(BE) ~ R-C-R1(BE) 6Ij I6 P6 81 7. 2R ~ direct process ~ R-R
9j 101 Scheme I Notes 11il R is a monomer unit. The bi or multifunctional 12l cross-linking reagent of formula I is designated in each of 13 the reaction~. One of the reactive groups (A) of formula I
14il ha~ been specified for each reaction. The other reactive group~ (B, E) of formula I may be cho~en from any of the 16 designation~ indicated for A of reactions 1~7 or one of (B, 17 ! E) may be hydrogen. Homo and hetero bi-functional and 18l multifunctional reagents are included. R1 o~ formula I and 19ll formula P of each of the reactions is an aliphatic group o~
20 j at least 2 carbons or an aromatic group. Preferably, R1 is 21i an alkylenyl group, -(CH2)j-; or an acyl group, -(CH2)k~
2~l C6H4-(CH2)1 - wherein j ls about 2 to about 30, and k and 1 231~ independently are O to about 20.

~9 l ll 3~
Il , ~L~3~36~
;

1 Table 1 2~ . X groups include:
halide, especially Cl, Br, I
2,4-dinitrophenoxy (an activated ester) p toluene ~ulfonyloxy 6 pivaloyloxy , t-butoxycarbonyloxy acetoxy.

2. Y groups include:
ll Cl, Br~ I.

3. X' groups include:
14 halide, expecially Clt Br, I

2,4-dinitrophenoxy(an activated ester) . pivaloyloxy 17 t-butoxycarbonyloxy acetoxy, 20: ;

~, , 2~

,. :

. -34- .
. , , ~ :373 ., The monomer units may be any enzyme which will react 2ij ~I with an appropriate qubstrate to produce a colored, Il ~luorescent, lumine~cent, electron den~e or radioactive 4 l product. Also 9 the en~yme may react with a colored, fluore~cent or luminescent sub~trate and quench it. The production or quenching of color, fluorescence or lumine~cence i may result ~rom direct enzyme cataly~is or the enzyme may produce an intermediate which enter~ into a chain o~ reaction~
to produce or quench color ~luorescence or lumine~cence.
If an electron dense or radioactive substrate is to be used, the enzyme will act to immobilize it. This may be accompliqhed by rendering the substrate insoluble, chemically r~active toward the enzyme or otherwi~e generating an immobilizing physical characteristic. With thi~ type of vi~ualization polymer~ the quantity of radioactivity immobilized by the enzymatic reaction or an electron ~ micro3copy determination of the quantity of electron den~e 18 material present will allow analysis of the minute quantity o~

target. Examples of ~uch enzymes include peroxidase, alkaline or acidic pho~phata~e, galactosida e~ gluco~e oxidase, NADPase9 lucifera~e, carboxypeptidase and the like.

The monomer units may also be natural or ~ynthetic ~3 polypeptides, polyol~, polyolefin~ or carbohydrates which are 24 ,1 ! ~ tagged. These may be based upon a polyamide backbone, a l polyether backbone, a polyvinyl backbone, or poly (sugar) 26 i ~ backbone. For the polyamide 9 the amino acid or diamine compound and dicarboxylic acid compound u~ed to make the backbone may be nonfunctional~ i.e., composed of a methylene unit chain ending in the appropriate functional groups, or it l may be ~ubstituted with group3 which would provide Qide chain . ~ !

, .

373~

i .

1 functionality. Examples would include glycine, alanine/
serine, lysine, aQpartic acid and the like a~ amino acidQ.
Example~ of diacids and diamine~ include arylene or alkylene ' dicarboxylic acid having at least 6 car~ons in the arylene group or 1 to 20 carbons in the alkylene group, and arylene or alkylene diamineq having at leaqt 6 carbons in the arylene group and 1 to 20 carbon~ in the alkylene group. ExampleY
will include poly(3-aminopropionic acid), polyglycine poly(glycyl-lysine), poly(N-(aminohexyl)alipic amide), poly(N-(aminobutyl)terephthalamide~ and the likeO
11 For the polyethers, epoxide~ and/or oxacyclic 12 compound~ with or without hydroxyl substitution can be used as backbone building blocks. Acidic condensation will couple the oxide compound~. Also, the polyols may have a poly(vinyl) backbone with hydroxylic subQtitution. These may be formed by vinyl/free radical polymerization of alkyl alcohol, butene diol and the like.
18 For the polyvinyls~ vinyl compounds with or without chemical group substitution may be uqed a~ backbone building blocksO Vinyl/free radical polymerization of such compoundQ-as acrylamide, acrylic acid, maleic aeid, alkyl ~ulfide, acrylonitrile~ methyl acrylate9 hydroxyethyl acrylate, alkenyl amine, acrolein, etc.-will produce the polyolefin 24 ' ; monomerq.
Il For the poly(~ugar), glycoqidic linking through hemi-ketal condensation of simple ~ugar building blocks can be u~ed a~ the carbohydrate backbone formation proce~sO
2~
Carbohydrates such as methoxy cellulose, poly(gluco e) starch, dextran9 polymaltose, amylose, etc. are examples.
~ The chemical tagY include the known, colored t ., j -36-I 1 ~23~

.
! fluoreqcent, luminescent, radioactive and electron den3e probe~ which will chemically bond with subqtituents pre~ent in a natural or synthetic polypeptide~ polyols9 polyolefin~ and i carbohydrates. These include probes with carboxylic acid derivative ~ubstituents, sulfonic acid ~ubstituent~, imino e~ter qubqtituent~, maleimide substituents, aldehyde qubstituent~9 azide ~ub~tituent~ and amine subqtituents which will react with the appropriate functional group of the monomer as outlined in Scheme I and Table 1. The probes will 10;
be monofunctional rather than difunctional ~o that they may 11 ' react only once with a monomer chemical group or backbone moiety. Examples of color tagQ include azido indigo dye, and congo red with sulfonyl chloride qubstitution. Example~ of 14~ ;
fluore~cent tag~ include fluorescein with an azido or 3ulfonyl chloride reactive sub~tituent, 3-azido-(2,7)-naphthalene 16;
di~ulfonate and rhodamine. Examples of radioactive tag~
include wood reagent (methyl p-hydroxybenzimidate) HCl which can be iodinated, and p-iodobenzenesulfonyl chloride.

Example~ of electron dense tag~ include collodial gold, colloidal ~ilver, ferritin, metal binding protein~ and ` reactive lead ~altq.

I olation and purification of the vi~ualization polymer of the invention may be accomplished by known 24!j techniques used for polymer i~olation~. Thes0 include ~5,' ~ dialyzation, lyophilization, chromatography, electrophore~

centrifugation, precipitation by electrolyte adju~tment or qolvent lipophilicity and the like.
2~ ;
~he carrying arrangement of vi~ualization polymer 29' i and detecting agent may be direct or indirect. The direct carrying arrangement will have the detecting agent covalently !
I

~L237~9 ;

., 1 `
bonded to the visualization polymer by a bifunctional or 2!i multifunctional cross~linking reagent. Generally, the bonding will follow Scheme I and method given for linking the monomer units of the polymer. These methodQ are generally known; for example see K. Peters? et. al., Ann Rev. Biochem., 46, 523-551 (1977); F. Wold, "Method3 In Enzymology XXVn~ pp 623-651 (1972) or M. Das, et al., Ann Rev. Biophys. Bioeng., 8 165-193 (1979). As with the visualization polymer, covalent linkage with chemical groups or backbone moieties of the detecting I agent should take place in a region of the agent which will not interfere with its ability to detect the target. This may be determined by any of the methods given above, especially the emperic method.

The indirect carrying arrangement may be of two types. In the first, the detecting agent may be multivalent and have an affinity for the visualization polymer as well as ~ the target. For example, it may be accomplished by employing a multivalent antibody which croqs-reacts with the monomer units of the visualization polymer and by utilizing the i appropriate amount of antibody and polymer so that at least one of the affinity site~ of the antibody remains open. The visualization polymer may also be bonded to a ligand which complexe~ with a multivalent ~etecting agent. Thi~ will 24 , ll accomplish the same kind of carrying arrangement.
25 ' In the ~econd type o~ indirect carrying arrangement, 26 ~
there will be an intermediate ligand binding compound intersper~ed between the detecting agent and the visualization polymer. It will display a high a~finity for specific ligands ' and will include an antibody, lectir, avidin, 3treptavidin, a - ~ DNA repressor protein, a high affinity enzyme7 a sequence !

!, I
i _38~

~:3~369 . I , .

specific polynucleotide binding protein or a complementary polynucleotide sequenGe. The agent and polymer will be 3 !
corre~pondingly labelled with the appropriate ligand. The ~1 i ligand may be ~oined to the detecting agent and polymer through a linker similar to a bi or ~ultifunctional cross-linking reagent R1(ABE) o~ Scheme I. Attachment of the linker ! to the agent and polymer will follow the methodQ given ~or the

8 1 i reagent coupling according to Scheme I. Also, tha ligand may

9' be substituted ~or a reactive group of the bi or

10 ' multifunctional cross-linking reagent R (ABE) of Scheme I.

11;
' Alternatively, the ligand may be covalently bonded

12 directly to the detecting agent and polymer. That i~ the

13 ligand may be bonded to a chemical group of the polymer and

14 detecting agent which may include an amine group, mercaptan group, carboxylic acid group, hydroxy group, aldehyde group or a C-H eroup. The procedure~ and reagents given according to Scheme I will be used for thi~ purpose and the appropriate reaction will be chosen depending upon the kind of reactive 19` ' group present on the ligand.

Methods for the preparation of the carrying arrangements and complexes o~ the invention follow the well ~ known procedures given in the foregoing background. Examples I include use of ligand~ ~uch as biotin, iminobiotin, poly-24~l i~ nucleotide ~equence~, enzyme ~ub~tratesg sugar3, haptene~ ~uch 25 l a~ 2,4-dinitrophenol, 2,4-dinitrophenylalkylcarboxylic acid 26~l having from 1 to 20 carbons in the alkyl group~ and carboxylic acid derivative~ thereo~. Other examples of haptene~ include l 2,4-dinltrophenylalkylamine havlng from I to 20 carbonq in the alkyl, phenylar~enate9 inistol and trinetroben~ene.
A preferred example of this type of carrying ., , ! i _39_ i, ~ 3'73~9 !

1' arrangement and complex is based upon use of a complementary strand of polynucleotide aq a detecting agent for a specific native polynucleotide sequence. Avidir. or streptavidin is used as the ligand binding compound and a functional~zed ; biotin or imino biotin derivative is used a~ the ligand.
Bonding the biotin or imino biotin to the visualization polymer and polynucleotide detecting agent may be accomplished directly or through use of a linker group. These methodq are known in the art; see Langer et al., Proc. Nat'l. Acad. Sci.
lo USA, 78, 663~-7 (1981); and follow the methods given for ll Scheme I except that one end of the bifunctional cross-linking 12 reagent will have been reacted with biotin or iminobiotin.

Accordingly, the complex includes avidin or streptavidin-(biotin or iminobiotin ligand)- visualization polymer. The carrying arrangement in addition includes the biotin or imino biotin labelled polynucleotide detectlng agent.

The method of the invention utilizing this example can be practiced as follows. An isolated double strand of 19 ' , , native polynucleotide to be detected, such as viral DNA, i~

broken or nicked with a DNAase at random points along each strand. Labelled nucleotide monomers are then translated into tha nicks u~ing a polymerase enzyme and the other associated ; strand as a template. Alternatively, the complementary 24 l l~ strand~ can be directly labelled with biotin label. Th~

labelled complementary pair of polynucleotide strands are then ~6 denatured and mixed with a denatured mixture of unknown native polynucleotide~, su~pected as containing the polynucleotide to be detected. I~ it i~ preqent, hybridization will occur and the labelled double strand may be visualized with the polymer complex.

--4~--"
36~ 1 .

A ~econd preferred example of a complex is derived i ~rom the methods given in the Backgrvund for PAP or ABC
complex methods or according to Langer et al., qupra. In this ; example, avidin or streptavidin is used a~ the intermediate ligand, an antibody, lectin, or a sequence speci~ic polynucleotide binding protein is used as the detecting agent and a biotin or imino biotin compound i~ used as the ligand complexing the visualization polymer and detecting agent with avidin or streptavidin.

In either o~ the~e two preferred examples, the 11 biotin or imino biotin compound may be directly coupled with amine or hydroxy groups of the polymer and agent through the use of amide bond or ester bond forming coupling reagents respectively. These methods follow reactions 1 and 5 of Scheme I. It may al~o be coupled through a linker group quch as that de~cribed above. The linker group i9 similar to the 17 bifunctional cross-linking reagent (R1ABE) o~ Soheme I except that one of the two reactive group~ will be an amine or lg acylhydrazide group wh~ch is coupled with biotin or ~0 iminobiotin. I
The visualization polymer oP the present invention may be used to detect minute quantities of target molecules.

These molecule~ may be found in biological material such as i tissue and fluid a3 well as in artificial or synthetic ~y~tems. Example3 include blood, lymph, urine~ feces, orga~
ti~3ue ~uch aq lung, liver, skin; kidney and the like 7 microorgani3ms, plant ti~sue, cultured cells~ hybrid cells, cells with recombinant DNA, synthetic mixtures of polypeptide~, immobilized enzyme sy~tems, synthe~ized DNA and ; other biological material.
~41-j: .

3~73~

:

1`
The target molecules may constitute any inorganic or organic species which i~ capable of producing an affinity with a detecting agent. Preferred targets will be found in the forego}ng biological material and systems. Example~ include S
proteins, lipids, carbohydrates, pho~pholipid~, fats9 nucleotides, nucleosides, nucleo~ide base~ polynucleotides, polypeptides, cancerogenic agent~, drugs, antibiotics, pharmaceutical agents, controlled substances, polymers, silicone~ organometallic compounds, heavy metals, metal-10.
protein complexes, toxic inorganic salts, and other agents or compounds produced by or having an effect upon a biological organism or material derived therefrom.

Generally, the procedureq for combination and, incubation of the detecting agents and targets are well known.

They follow method~ used for affinity and immumodiagnostics assay~; see for example L.A. Sternbeyer, "Immunohi~to-chemistry" cited above. For example, combination of metered amounts of agent and target in buffered aqueous solution lg folIowed by incubation at temperature~ from ambient to about 37C for periods such as 5 minutes to 18 hr. will cause con~ugation. Addition of the visualization polymer or itq complex under similar conditions will then provide 23;
; visualization. Finally, if the agent is bonded to the , vi~ualization polymer, similar technique~ can be followed~
U~e of the visualization polymer for the ~oregoing 26 ;' 27 detection purpose~ ha~ advantage since it allow~ detection of extremely minute quantitie~ of target molecules. It may be employed in medical diagno~tic laboratory a~ an analytical technique for identification of biological products in fluids and tis~ue~ which are indicative of a disease state or ~3~3~i~
;

malcondition. These would include for example, abnormal amounts of growth hormone, the presence of human gonadotropin 3;
indicating cancer, detection of riral invasion, quantification of hormone and regulatory enzyme levels. Al~o, it may be employed to perform normal ~luid and tissue chemistry analyses and may be employed in the biochemical research laboratory as a tool for identification of biochemical substances.
The visualization polymer may be used in synthetic protein or polynucleotide work to id~ntify synthesi7ed, semi-synthetic or native proteins and synthesized~ recombinant or native polynucleotides. Applications will be found in the course of preparative or bulk work to produce useful proteins such as inYulin, interferon, ACTH, gonadotropin, oxytocin, pituitary hormone, LH, FSH and the like by such techniques as recombinant DNA or hybridomas.

The carrying arrangement of detecting agent and visualization polymer complex will be the form for u~e to perform the foregoing analyses. Since the polymer will provide multiple signals from the carrying arrangement association with the target, chemical amplification will result. In the pre~erred form of the carrying arrangement wherein a complex of polymer and ligand binding compound is employed, the signal amplification by the polymer will be further increased by multivalent liganding of multiple number~

of polymer to each molecule of detecting agent. Aocordingly, in the preferred embodiments employing an antibody or complementary polynucleotide qequence detecting agent, biotin or immobiotin labels 9 on the agent and polymer9 and an avidin or streptavidin, detection of femtomole (10 ) quantitites can be achieved. This will also depend in part upon employing ~D Ir3 a~
, i a sensitive monomer unit system and the appropriate carbon 2 ' chain linker lengths for both the biotin ~abels and the coupling agent of the polymer. An example would be use of the enzymes alkaline pho~phatase or horseradish peroxidase coupled as visualiæation polymer by epsilon amino group bonding with an active diacyl derivative of suberic acid, and use of biotin labels with carbon chain linkers of from 6 to 14 carbon in length.
The polymer, complex and carrying arrangement of the invention may be formulated as an integral part of a solid or liquid detection system and kit. Colorimetric, fluorescent, lumine3cent and radioactive systems may be prepared in this manner. Such systems and kits would include the detecting 14 components, i.e., the polymer, its complex with a ligand, a ligand binding compound, and the detecting agent as well as 16 the appropriate chemicals, reagents and ~olutions in metered 17 amounts and standardized concentrations al~o. For example7 if enzymatïc action with a ~ubstrate to produce a colored product is to be the visualization procedure employin~ the polymer, the system and kit will contain the chemicals, substrate and reagents necessary ~or performing this analy~i~. These materials will be present as metered quantities so that the light absorption produced by the colored product may be used in con~unction with a st~ndard Beer's Law mathematical formula to determine the concentration of target detected. Usually, a standard reaction o~ polymer with sub~trate will be employed as a control and reference, although standard graphs o~

ab~orption relative to concentration may al~o be utilized.

Fluorimetric, lumime~ric and radiometric analyses may be performed in a similar fashion. The intensity of 1~3~3~9 .

fluorescence, luminescence or radioactivity produced by the polymer in the carrying arrangement associated with the target - i will be measured by the appropriate electronic machine.
4' Necessary reagents and chemicals will also be present.
Metered amounts of component~ will be employed qo that the intensity value may be corralated with the quantity of target using a standard Beer'~ Law mathematical` ~ormula.

In these systems, a concentration of detecting agent-visualization polymer complex will be used in the test solution which is sufficient to associate with all the target to be detected. Preferably, the concentration will provide an 12 exces~ amount. The target may be grossly separated from other material by sedimentation9 by centrifugation, or otherwise separated by such techniques as high pressure liquid chromatography, gel permeation chromatography, electrophoresis, precipitation, thin layer chromatography, paper chromatography or ~imilar techniques. However 9 this is not necessary for the purposes of this invention. The signal producing reaction will be initiated by forming the target-detecting agent conjugate followed by forming the visualization polymer-detecting agent associative arrangement and measuring the visualization signal from this arrangement.

Comparison of the signal intensity with a standard graph will yield the quantity of target. Other techniques such a~

con~ugate-complex exchange, which are known in the field of immunoanalysis, may al~o be used.

With all of the foregoing liquid and solid analysi3 methods, qualitative detection may also be ~ade. Since this object will be determination o~ the presence of the target to be detected rather than quantity) standardization need not be I

!
~;~3~7369 I i , .

used. The qualitative technique~ will generally follow the methodq ~or the foregoing quantitative techniques.
The invention will now be ~urther illu~trated 4~ ' i through the aid of example~. These example~ are not limiting and other similar procedure~ as ~hown by the examples will be readily apparent to those ~killed in the art. All measurements are provided in the metric system unless otherwise noted.
EXAMPLES

Abbreviation~ and acronym~ u~ed in the exampleq are ~1 defined as follow~.

13 Bio-4-dUTP, Bio-ll-dUTP and Bio-16-dUTP; analogs of TTP that contain a biotin molecule linked to the C-5 position of the pyrimidine ring through linker arms that are 4,11 and 16 atom~ long, respectively.
16 Bio-4-DNA, Bio~ DNA and Bio-16-DNA are DNA probes prepared with Bio~4, Bio-11 or Bio-16 dUTP analogq, respectively.

DNA is poly(deoxyribonuoleic acid).
IgG i~ immunoglobulin G fraction.

DSS is disuccinimidyl ~uberate.

BACSE is biotinyl-epsilon-amino caproic acid N-hydroxy~uccinimide e~ter.
24 i ! NMZT buffer i~ defined at p I~R.

poly ABAP Complex i~ avidin-biotinylated alkaline pho~phata~e polymer complex DAB i~ 3,3-diaminobenzidene.

! EAC i~ ethyl aminocarbazole.

The following example~ ~how the ~ynthe~i~ of vi~ualization polymer~ of inte~tinal alkaline pho~phata~e and ; :

37~
;
i the construotion of avidin (or streptavidin) enzyme polymer complexes. The~e polymers provide visualization which i~ 20-,~ 50 fold ~ore Qensitive than heretofore known immunologic or affinity reagents. Also deqcribed are rapid and senqiti~e procedureq for visualizing biotin-labeled DNA probes a~ter hybridization to target sequences immobilized on nitrocellulose filters and methods for detecting protein antigenq in tissue sectlons a~ter application to glass qlideQ.
g The examples and procedures chosen illustrate application of the invention to detection of a genetic sequence a~sociated with human placental DNA and a peptide hormone associated with malignancy. These are the alpha and beta globin genes in human placental DNA and human chorionic gonadotropin. Analysis o~ human genes can be uqed for prenatal diagnosis of genetic disorders and analysis o~ hCGT

can be used as an oncofetal marker of cancer.

Affinity purified rabbit anti-biotin IgG was prepared as described Langer-Safer, Proc. Natl. Acad. Sci.

USA, 79, 4381 (19B2). An ammonium ~ulfate fraction of goat anti-biotin IgG was provided by Enzo Biochem Inc., NY.

Biotinylated rabbit anti-goat IgG9 biotinylated goat anti rabbit IgG, and avidin DH - biotinylated horseradiQh peroxidase complex (Vectastain ABC kit) were purchased, or 24 i provided a~ gifts, from Vector Laboratories,Inc~, Burlingame, CA~ Streptavidin, Hoffman, Proc Natl. Acad. Sci. USA~ 77, 4666 (1980), was obtained from either Bethesda Research Laboratories, Inc.~ Gaithersberg, MD or Enzo Biochem Inc~

Calf intestinal alkal~ne phosphata3e (Cat. No. 567 752) and pancre~tic DNAse type I were purchased from Boehringer Mannheim, Indianapoli~, Ind~ Biotinyl-ep~ilon-amino caproic ~:3~3~

acid N~hydroxysuccinimide ester was synthesized according to 2 '.
; Costello et. al., Clin. Chem., 25, 1572 (1879).
Disuccinimidyl suberate was a product of Pierce Chemical Co., Rock~ord 9 Ill. Re~triction endonuclea~es and E. coli DNA
polymerase I were obtained from New England Biolabs, Ino., Beverly, MA. Agaro e (type II), bovine ~erum albumin (BSA, fraction V), 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt, cadaverine free base, 3,3'-diaminobenzidine tetra~ydrochloride, ficoll (type 400~, ethyl aminocarbazole, herring sperm DNA (type VII), nitro blue tetrazolium (grade III), and polyvinyl pyrrolidone (PVP-40) were ~rom Sigma Chemical Co., St. Louis, M0. Plasmids containing human globin gene sequences were provided by Dr. Sherman Weissman, Yale Univerqity JW101 is a 0.4 kbp cDNA alpha_globin clone and pHb C6 is a 5.2 kbp genomic ~ragment containing the beta-globin gene in pBR322, Fukumaki, Cell, 28, 585 (19B2), Plasmid pMM984 contains the complete 5.1 kb genome of the parvovirus, minute virus of mice (MVM), cloned into-pBR322.

This plasmid contains a single Xho I site within the MVM
sequence insert. Human placental DNA was a gi~t from Scott Van Arsdell 9 Yale University.
Polymerization and Biotinylation of 23 Intestinal Alkaline Pho~phata~e 24 i Calf intestinal alkaline phosphatase was polymerized by cross-linking with disuccinimidyl ~uberate (DSS). The 26 ! enzyme, commercially ~upplied as a 10 mg/ml solution, was 27 diluted to 1 mg/ml in ice cold N~ZT buffer (3 M NaCl, 1 mM
28 MgC12, 0.1 mM ZnC12~ 30 nM triethanolamine, pH 7~6) in a 29 silanized glass reaction vessel or a silanized Eppendorf tube.
All subsequent reactionY were done at 4C~ A 5 mgJml solut~on 48~
.

~3~36~3 1' of DSS in dimethylformamide was added (10 microl/ml of enzyme ~olution) in two equal aliquots ( 30-60 seconds apart) with ' gentle stirring. This soIution, containing an 19-fold molar 4 ' .
exces~ of DSS over enzyme 9 was stirred for 20 minuteq during which time a coudy precipitate appeared. 10 microl aliquots of cadaverine free base (0.1 mg/ml in NMZT bu~fer) were added to the reaction mixture four times at 10 minute intervals.
The DSS-cadaverine ratio was then made equimolar by adding g another 30 microl of cadaverine solution for each ml of ~ reaction. After stirring an additional 10 minutes, 2 microl ll of undiluted cadaverine-free base qtock was added and the mixture stirred a further 30 minutes. The resultant clear solution was then dialyzed extensively against NMZT buffer.

(The polymerization and cadaverine treatmentq were often repeated a second time, however these additional steps did not appear to increase the polymer size significantly since both lX and 2X polymerized enzyme complexes detect target sequence~

with about equal sensitivity.) Monomeric or polymeric forms of alkaline phosphatase were reacted with a 60-fold molar excess o~ biotinyl-epsilon-amino caproic acid N-hydroxy~uccinimide ester (BACSE) by adding 10 microl. of a 20 mg/ml solution of BACSE (in dimethylformamide~ for each mg of enzyme present in the dialysis bag. After agitating the bag on a rotary shaker at 25 ~
4C for 2 hourq, the reaction mixture was again exten~ively dialyzed againqt NMZT buf~er. Sodium azide was added to a Pinal concentration of 0.02% (W/V) and the biotinylated enzymeq stored at 4C until use.

~49~

~ 3'73~9 Preparation of Avidin-biotinylated Alkaline 2 ' Phosphatase PolYmer Complexes ~Poly ABAP Complexes) 3 , The biotinylated enzyme polymer was mixed with a 4 slight excess of avidin to produce complexes capable of direct interaction with biotinylated probes hybridized on filters.
6 The protein mixture that gave an optimal signal to noiqe ratio 7 was determined emperically by analyzing the ability of various 8 protein mixtures to discriminate between avidin-DH and g biotinylated goat IgG spotted on nitrocellulose. Protein ratios were ad~usted to give a strong reaction with 11 biotinylated IgG but little9 if any, signal from the avidin-DH
12 spot. The component composition of the poly ABAP complex was 13 further optimized for high ~ensitivity and ~pecificity against 14 Bio-16-DNA samples spotted on nitrocellulose, using a constant avidin-DH concentration and varying amounts of biotinylated 16 enzyme polymer. The optimum protein ratios were established 17 for each new lot of enzyme polymer to ensure maximum 18 specificity and sensitivity. En~yme complexes were made using 19 avidin-D~ or streptavidin and complexe~ made with either biotin-binding protein gave similar reqults.
21 The poly ABAP complex used was synthesized as 22 follows: Avidin-DH (1.ô mg/ml) was diluted to a concentration 2~ of 7.2 microg.~ml into AP 7.5 buffer (0.1 M Tris-HCl, pH 7.5, 24 ~ 0.1 M NaCl, 2 mM MgC12, 0.05% (V/V~ Triton X-100). The protein was added to a elean boro~ilicate gla~s tube prerinsed ~6 with a solution of bovine serum aLbumin (3%, W/V~ in AP 7.5 27 buffer. Biotinylated alkaline phosphatase polymer (0.92 mg/ml 28 in NMZT buffer) was then added to a final concentration of 1.8 29 microg/ml and complex formation allowed to proceed for at least 10 min prior to u~e.

l ) l .~3~369 .....

2 Example 1 Preparation of Dot Blot3 Plasmid pMM984 DNA, either linearized by Xho I
digestlon or nick-tran~lated with Bio-11 or Bio-16-dUTP
sub~trate wais ~erially diluted in 50 mM Tris:Cl, pH 7.5, containing 0.3N NaOH and 1.5 mg/ml of qhearcd herring ~perm DNA. Appropriate dilution~ were neutralized on ice with ice cold 3 N HCl and 5 microl. aliquots spotted direc~ly on BA-85 nitrocellulose filter sheet~ (Schleicher and Scheull, Keene, NH) on top of Saran*wrap. Filters were air dried 9 baked for 4 hr at 80-C, and cut into strips containing a dilution ~eries ranging from O to 128 pg of plasmid DNA per ~pot.

Agarose gels approximate1y 6 mm thick were prepared on a horizontal electrophore~i~ apparatus and DNA ~amples were electrophoresed a3 dei3cribed by Alwine, et. al., Methods Enzymol., 68, 220 (1980). DNA wa~ transferred to nitrocelluloi~,e sheetA (BA-85 or Sartoriu~ 11336) preqaturated with 20X NaCl~citrate as de~cribed by Southern and modified by Thomas, J. Mol. Biol, 98, 503 (1975); Proc. Nat. Acad. Sci.

USA, 77, 5201 (1980), after per~orming the acld depurination ~tep of Wahl, et. al., Proc. Nat. Acad. Sci. USA, 769 3683 (1979). Complete tran~er was en~ured by using a 3 cm thick 24;
~ ~oam qponge s,aturated with 20X NaCl/Cit a~ a fluid rei3ervolr beneath the gel. DNA filteris were air dried, baked at 80-C
26 ;
for 2-4 hr and stored at 4-C over CaS04 untll hybridized.

i PrsbeiY were prepared by nick trani~ilation eisi~entiaIly a~ deis,cribed by Rigby et al., J Mol. Biol., 113~ 237 ~1977).

Iisolate~ of double stranded DNA to be detected were nick , * Trade Mark translated with E. Coli DNA polymerase I which catalyzes the coupling reaction of nucleotide residues to the 3' hydroxy 3 , , terminu~ of a nick or break in the DNA ~trand while ; eliminating nucleotide residue~ from the 5' phosphoryl termini. The complementary DNA strand served as a template.
Thi~ procedure allowed addition of labelled nucleotide re~idue3 to the double DNA strand. The strand was then denatured and each ~equence served as a complement for g hybridization with the native DNA strand partners in an I unknown mixture.

For thi~ process, reaction mixtures (20-200 microl.) contained 40 microg/ml DNA, 200 units/ml DNA

polymeraqe I, 0.005 microg/ml DNAase (nicking snzyme), 50 mM

Triq-Cl pH 7.5, 5 mM MgCl2, 50 microg/ml BSA, 50 microm.
each dCTP, dGTP, dATP and TTP (P-L Biochemicals, Inc., Milwaukee, WN) or Bio-dUTP analog~. Bio-4-dUTP, Bio-ll-dUTP

and Bio-16-dUTP were synthesized by method~ according to Langer, Proo. Nat. Acad. Sci. USA, 78p 6633 (1981). Nick-translation reactions containing Bio-dUTP analogs were incubated for 2 hr at 14C while reactions with TTP were incubated for 1 hr at 14C. Some of the Bio-dUTP reactions also contained 200 Ci/ml of alpha- P-dCTP tAmersham~ 410 Ci/mmole~. High ~pecific radioactivity probe~ were prepared a~ above wlth TTP and alpha P-dATP at 5 microm and 1 ooa Ci/mmole (Amer~ham).

Prehybridization and Hybridization Condition~

The general protocol outlined below was ~ound to be optimal when u~ing biotin-labeled hybridization probe3.

Filters were prehybridized ~or 2-4 hour~ at 42C according to Wahl et. al., Proc. Natl. Acad. Sci. USA, 76, 3683 (1979) in a 1~73~ 1 sealed plastic bag using a mixture containing 45% (V/V) deionized formamide (conductivity 10 MH0, pH 6.5 or le~s), 5XNaCl Cit Denhardt's solution; Biochem. Biophys. Res. Comm., 23, ~41 (1966)~ 25 mM NaP04 buffer, pH ~.5, and ~onicated herring sperm DNA (250-500 microg/ml). The hybridization buffer contained 45~ (V/V) formamide, 5XNACl Cit, lX
Denhardt's qolution, 25 mM NaP04 buffer, pH 6.5, 10% dexSran sulfate (added as 20% of the final volume from a 50% (W/V) stock), 250-500 g/ml sonicated herring sperm DNA and 200-500 ng/ml of a DNA probe that had been nick translated with Bio-ll 16-dUTP. The carrier and probe DNAs were heat denatured ~ust prior to addition. 10 ml of hybridi~ation mixture was used per 100 cm of filter and the hybridization reaction incubated at 42C (in a sealed bag) for 30-180 minute~. For analysis of single-copy mammalian gene sequences, hybridization was 67 generally done to a Cot of 0.8 x 10 2 (e.g., for a probe concentration of 350 ng/ml the hybridization time wa~ 120 minutes. Following the hybridization, filters were washed 4 time3 at room temperature, 2-3 min for each wash, twice with 2XNaCl/Cit - 0.1~ NaDodS04 and twice with 0.2X NaCl/Cit - 0.1%

NaDodS0~. Two stringent washes (15 min each) were done with 0.16XNaCl/Cit - 0.1% N~DodS04 at 50C, followed by two washes at room temperature. Filters were air dried, and mounted for autoradiography with XAR X ray film (Kodak, Rochester, NY) or as~ayed for biotin as de~cribed below.

27 Colorim0tri Detection of Bio-DNA Probe~
Dry nitrocellulose filter blotq were incubated at 2~
42C for 15 min in a 3~ (W/V) solution of bovine serum albumin (BSA) in AP 7.5 buffer, air dried, baked at 80C for 30-60 min, and then rehydrated in the BSA - AP 7O5 buffer at 42~C

~L~373~9 !

for 20-30 min. Small filter strips were treated in 13 X 100 mm glass tubes while larger sheets were treated in ~ polyethylene trays or heat-sealed polypropylene bag~. Filters '! were exposed to enzyme complexes for 5 min at room temperature; 2-5 ml of complex were used for each 100 om2 of filter. Filters were rapidly washed 3 timeQ in 250 ml of AP
7.5 and twice in AP 9.5 (0.1 M Tris-HCl, pH 9.5, 0.1 M NaC1, 5 mM MgG12). When avidin peroxidase (ABC) complexes were used 2X NaCl~Cit was substituted for AP 7.5 and AP 9.5 buffers.

For development with ABAP or poly ABAP complexes, filters were inoubated at room temperature for 0.5 to 24 hours in AP 9.5 buffer containing 0.33 mg/ml of NBT and 0.17 mg/ml of BCIP; see Histochemie, 23, 1806 (1970). The phosphatase 14 ~
substrate solution was prepared as follows: for each 15 ml or reagent, 5 mg of NBT was suspended in 1.5 ml of AP 9.5 in a microcentrifuge tube and vortexed vigorously for 1-2 min, centrifuged briefly in a micro~uge, and the supernatent decanted into 10 ml of AP 9.5 warmed to 35 in a polypropylene 19 .
tube. The residual NBT pellet was extracted twice rnore with 1.5 ml of AP 9.5 buffer and the supernatents pooled with the original solution. The tube was rinsed with a final 0.5 ml of AP 9.5 that was also decanted into the 15 ml NBT stock solution. BCIP (2.5 mg) was dissolved in 50 micro of N~N-24 l ` dlmethylformamide and added dropwise with gentle mixing inko 25 i !
~ the NBT solution~ Care should be taken not vortex or26 ' vigorously shake the reagent mixture since this can lead to the formation of undesirable precipitates. Although this reagent wa~ generally prepared fresh, it was stable at room temperature f~r at least 24 hour~ in the dark.
Filters were incubated with the ~ubstrate solution -54~

in sealed polypropylene bags, using t o- 15 mls of ~olution/100 ~m2 of filter. To reduce nonspecific background, color Il development should proceed in the dark or subdued light.
4 j Single-copy mammalian gene sequences generally become visible withln 30 mlnutes, although highly colored hybridizatlon signals require incubation times of several hours. Color development was terminated by washing filters in 10 mM Tris-8~
HCl, 1 mM EDTA pH 7.5. Developed blots were ~tored dry or in ~ heat-sealed bags contalning a small amount of 20 mM Tris-HCl, 5 mM EDTA, pH 9.5. Although the color intensity fades when the nitrocellulose sheet is dried, rewetting with buffer will restore color intensity a long as the ~ilter has been stored 13;
without prolonged exposure to strong light.

Using the foregoing procedure with a known amount of ; DNA to be detected, and standard immunological and affinity procedures ~or visualizing biotin-labeled DNA, the comparative ensitivity results were determined. Serial two-fold - dilutions of target (pMM984 plasmid) DNA, labeled with Bio-4-dUTP, Bio~ dUTP or Bio-16-dUTP, were mixed with a constant amount o~ carrier herring sperm DNA (7.5 microg) and spotted directly onto nitrocellulose strips. These strip were then incubated with various detector reagents and the sensitivity o~ each method determined. Results of such experiments are 24 . !
~ summarized in Table 2. Indirect lmmunofluorescence, using a 25 , i hand-held UV-light Cource for viqualization, wa~ a relatively ~6 insensitive procedure with detection limît~ near 1 ng of target DNA (Table 2 line t and 2). Indirect-immunop~roxidase methods, using either DAB or EAC as a sub~trate, were better (Table 2 lines 3 and 4), but ~till only 150-200 pg were seen with a Bio ll-DNA target. The peroxidase - antiperoxidase i~
j 1~3~ 9 I

ll! aqsay method of Sternberger; Sternberger et. al., J.

21 Histochem. Cytochem., 18, 315 (1970); improved the qensitivity i, . - . .
3¦ 2-fold over that seen with the indirect immunoperoxidase 4,'l technique. However it was alqo not ~ensitive enough ~or the analysis of single-copy mammalian DNA sequences. With each of 6 these immunological methodq, increasing the length of the 7l biotin "linker arm" from 4 to 11 atoms enhanced the 8jj detectability o~ the target by 4-fold. In contraqt, Bio-4-~; I
9, DNA was not detected at all by complexes of avidin and biotinylated horseradish peroxidase (the ABC complexes of HSU7 ll Hsu, et. al., J. Histochem. Cytochem., 29,. 577 (1981).
12 However, ABC complexes revealed Bio-ll-DNA and Bio-16-DNA with 13 equal e~ficiency and with a sensitivity limit of less than 100 14 pg in a simple one-step reaction (Table Z lines 6 and 7).
Complexes made with avidin-DH and biotinylated intestinal 16 alkaline phosphataqe (ABAP complexes) were even more sensitive l7 than ABC complexes made with peroxidase ~Table 2 line 10), 18 with detection limits between 20 and 30 pg of target DNA. All l9 attempts to increaqe the signal strength using multiple nsandwiching" techniqueq requlted in either no enhancement or 21 a sharp decrease in signal. Thiq was observed using either 22 antibiotin IgG or an ABC type complex as the primary detector 23, (Table 2, lines 8 and 9).
24l, As shown in Table 2, line 11, the intestinal alkaline phosphatase polymer of the invention synthesized 26 according to the foregoing procedure, which retained high 27 levels o~ enzymat~c activity in the complex, i3 termed poly , 28 ABAP. When used in con~unction with a substrate mixture of 29 nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, this complex detected 1-2 pg o~ target DNA with ,, 1, i, .
, -56- !

~Z3~
!

! I
! !

l enzyme incubations of 3 4 hours. This level of detection is 2 1~ t0n to ~i~teen-fold better than the moqt Qen~itive of the 3 1l other methods.
4 ''I Assuming 6 10 pg of DNA per diploid mammalian cell 5 j, an average "gene" size of 5 kbp, 12 pg qensitivity would be 6 ,I required for analyzing unique cellular sequences in a 7.5 microg sample of DNA. The poly ABAP complex of the invention ,I detected 1-2 pg of DNA and thus haq thiq capacit~.
9 ll Table 2 Relative Sensitivity of Biotin-Specific_Reagents in Detecting Biotin-labeled polynucleotides bound to Nitrocellulose Filters I Bio-DNA Detector Reagents Substrate3 Detection limits 13 Ij (pg target seq~uence) 14 ll Target .

15 , 1. Bio_4 Anti-biotin IgG+FITC-2o Ab - 2000 4000 Il 2. Bio-11 Anti-biotin IgG+FITC-2 Ab - 500-1000

16 ,l 4 Anti-Biotin IgG+HRP-2 Ab DAB/EAC 500-1000

17 ~l 4. Bio-11 Anti-Biotin IgG+HRP-2 Ab DAB/EAC 150-200

18 ! 5. Bio-4 ABC (avidin DH - Bio HRP) DAB/EAC None detect~d 6. Bio-11 ABC DAB/EAC 75-150

19 7- Bio_16 ABC DAB/EAC 75-150

20 ~ 8. Bio~16 Anti-biotin IgG~Bio-2 Ab~ABC DAB/EAC 100;200

21 ll 9. Bio-16 ABC~Bio-DNA+ABC DAB~EAC 100-200

22 ' 10- Bio-16 ABAP (avidin-Bio Alk. Phos.) NBT + BCIP 20-30 28 11. Bio-16 poly ABAP (a~idin~poly NBT ~ BCIP 1-2 ll Bio. Alk. Phos) 24 ,~1 `
25 '¦ Figure 1 qhows typical Dot Blot as~ay resultq 26 l, obtained uxing ABC, ABAP and poly ABAP enzyme detector 27 I complexe~ in the foregoing DNA detection method and 28 ¦ incubating 60 minute~ with known quantities of DNA to be 29 l detected. Peroxida~e (ABC) reactions (lane 1) generally gave 30 1, higher non-~pecific background on the nitrocellulo~e filter~
ii Il 57 ~ .

3~

. I
.
l I than ABAP or poly ABAP complexes (lanes 2 and 3, 2l re~pectively), particularly if incubations with peroxidaqe 3l sub~trates were longer than 30~60 minutes. However, u3ing the poly ABAP detector, 1 pg of target DNA ean be made Ij visible without significant background noise with enzyme 6 1,l incubation~ of 3-4 hours. Since the NBT/BCIP sub~trate ! mixture did not exhibit appreciable end-product inhibition of the pho~phatase activity~ the intensity of the signal could il be increased further by prolonging the substrate incubation ,I period for up to 24 hours.
Having developed an enzyme comple~ with the capacity to detect pg quantitities of biotin-labeled DNA, the optimum condition~ for u~ing Bio-11- or Bio-16-DNAs as hybridization probes were then inve~tigated. Two pMM 984 DNA
probes~ one labeled with 32p alone and the other labeled with 32p and biotin-nucleotides, and both being probe~ of identical ~peclfic radioactivity, were hybridi~ed at variouq probe concentrations ranging from 5 to 1000 ng/ml. The results at the variouq probe concentration~ are shown in I Figure 2, lines 1 6; the strip numbers at the top of each graph are target concentrations. The autoradiograph of the ~trips hybridized with 32P-labeled DNA showed significant non-specific background at all probe concentration~ above 25 ' 24',1 I ng~ml (Figure 2C 4 day3 expo3ure of strip to film). In 25l contra~t, the autoradiograph of the ~trip~ hybridized with 26' 32p labeled Bio-16-DNA probe gave virtually no non-3peci~ic 27~1 1 Il background noise at probe concentration3 up to 750 ng/ml ¦1 ~Figure 2B 4 day expo~ure of ~trip to ~ilm). Thi~ reqult ~9i 30¦ demon~trated that a good ~ignal to noi~e ratio could be l obtained u~ing very high concentration~ of a Bio-DNA probe, ll Il -58- 1 i~3~73 ,,.
ll l 1 ll thus making it possible to markedly reduce the hybridization times required to achieve any desired Cot value.
3 il The coloration signal ~rom the 3~P-labeled Bio-16-DNA probe visualized with the poly ABAP co~plex of the invention, which wa~ prepared according to the ~oregoing procedure, is shown (Figure 2A) to be more sensitive than the autoradîograph 2B and approximately equal to 2C. This development, however, only required 4.5 hr. to complete.
I Accordingly, the poly ABAP complex of the invention in this 1 , test is faster than known detection methods and has high . i 12 The ability of the poly ABAP detection system of the invention to visualize sequences in a Southern blot ~ormat was fir3t analyzed in a reconstruction experiment.
Various amounts of linearized pAT153 plasmid DNA were added i, to 10 microg. samples of sheared carrier DNA and the mixtureY
electrophoresed on a 1.4% agarose gel Figure 3A 3hows these 7 lanes o~ gel after electrophorsis and gel lane M as a marker which contained 0.6 microg o~ plasmid DNA stained with il ethidium bromide to indicate the location o~ the desired plasmid bands after electrophoresis. The regions o~ the 7 lanes o~ the gel containing the 3.6 kb plasmid bands were transferred to a nitrocellulose filter and the filter hybridized with a Bio-16-labeled pla~mid probe. To te~t critically the potential of the ~ystem, the hybridization was !I done under high stringency conditions (in 50% formamide) to a I¦ high Cot9 conditions known to give less than ~aximal result~

'I Ne~erthelesq, a~ter a two hour incuba~ion in the NBT/BCIP
2~ 11 j substrate solution the poly kBAB complex clearly detected il bands containing as little as 3.1 pg o~ plasmid DNA as shown il i Il -59- l 373~
ll l Il i .

., .
lll in Figure 3B. The intensity of the signal was al~o 2 ! proportional to the amount of target sequence.
3 ! Detection of Specific DNA in a Mixture of DNA'~
4~ ! The ability of the detector ~ystem of the invention to detect specific polynucleotide sequences in an unknown 6 mixture wa~ demonstrated by the following procedure. Human 7; placental DNA was digeQted with Eco RI, or Hind III and 8 transferred to a nitrocellulose filter after electrophoresis 9, in a 1~ agarose gel. The DNA was then hybridized for 2 hours with Bio-16-labeled alpha-globin or Bio-16-labeled beta-ll globin probes. These probes were complementary for the alpha 12 and beta globin genes (~NA) in the placental DNA. The 13 alpha-globin probe (clone JW101) was a cDNA clone that 14 contained only 400 nucleotides of the alpha-globin gene sequence; the beta-globin probe (pHB C6) was a 5.2 kb genomic 16 clone. Figure 4 shows the results. Lanes 1 and 3 are ECoR1 17 digestions, lanes 2 and 4 are Hind III digestion~, lane~ 1 18 and 2 are hybridized wit-h probes JW1019 lanes 3 and 4 are l9 hybridized with probe PHBC6. Lane 5 contained 750 pg each of lambda phage Hind III and PMM 984 PST1 digest~ in 10 microg 21 of sheared herring DNA and was hybridized with lambda and pMM
22 9~4 probe~. Restriction fragments were observed which had ~3 ~izes in good agreement with the published literature; see 24 Proc. Nat. Acad. Sci USA, 75, 5950 tl978); Cell~ 19, 947 25l ~198); Cell, 19, 959 (t980).
26, All lanes were visualized by polv ~BAP after 27 several hours of enzyme incubation a~ ~hown in Figure 4. The 28 minor 2.5 kb Eco Rl fragment hybridized to the beta-globin probe (Figure 4, lane 3) i5 most likely the Eco Rl fragment from the 5' region of the delta globin gene, which cro~s-hybridizes with beta-globin probe~ see Cell supra..
3 ¦ Although the three Hind III fragments that hybridized with i the alpha-globin probe (Figure 4, lane 2) exhibit a weaker 4 signal than the other bands observed in lanes 1, 3 and 4, ~¦ this is not surprising since each of these fragments hybridized to only a subQet of the 400 nucleotides present in the probe. It is clear, however, that unique mammalian gene ¦ sequence~ can be viqualized colorimetrically using the poly l ABAP detection system. Combining Bio-DNA probes with a poly ll ABAP detector Qystem thus provideQ a rapid and sensitive il non-iQotopic procedure for Southern, Northern or dot-blot 12 i l hybridization analysis.
13 1 Example 2 ll Compari~on of Monomeric and Polymeric Enzyme Yisualization 16 11 Polymers For Detecting Biotin-labeled Proteins 17l !! Proteins separated by gel electrophoresi~ can be -l, transferred to nitrocellulose filter sheets and specific proteins located by specific, labeled-antisera coupled with a lj biotin label detection system prepared according to the 21i~
!j method described in Proc~ Natl. Acad. Sci USA, 78, 6633 j (1981). Four enzyme systems for detecting biotin-labeled ! antibodies were compared.
24 Method 26l Biotlnylated goat anti-rabbit IgG (Vector i LaboratorieQ, Burlingame, CA) wa~ serially diluted in a carrier protein qolution of 0.5% w/v bovine serum albumin ~ (Sigma Chemical Co., St. Louis, M0) in 2X SSC. A serie~ of 8 i spots containing between 0.8 and 100 pg o~ labeled protein, 301 and one ~pot of carrier (biotin-free) protein ~olution were , -61-~i~3736 il , l,j placed on filter 3tripq. Filter~ were then baked at 80C for 2ll 1 hr, to fix the protein to the nitrocellulo~e ~trip.
311 Replicate strip~ were "blocked" by preincubating in 4l¦ 3% PSA in lX AP7.5 bu~fer or in 2% BSA in PBS with 0.1%
5I Triton X-100. Blocked ~trip~ were dried at 80C for 45 min., I. i 6 and rehydrated in blocking reagent for 30 min. at 42C. The 7ll amount of biotinylated protein ~target" that could be 8l detected by each of the four avldin: biotinylated enzyme 9l complexe~ was then a~ayed using the enzyme reaction~
lOIj ou~lined in Table 3.
ll, Table_3 12', Detector Complex Enzyme_ _ ~ rate Solution ~l l 13, 1. ABC kit (Vector/ hor~eradish 0.2 mg/ml amino 1 Laboratories) peroxida~e ethylcarbazole and 0.01%
}4j H 0 in 50 mM NaOAc ! b~f~er, pH 5Ø

~i 2. Detek II kit Same a~ 1 0.5 mg/ml diamino 16;; (~nzo Biochem) benzidine and 0.02~ H 0 17 in Enzo buffer. 2 2 3. ABAP calf 0.33 mg/ml Nitro Blue l8!l (monomeric) inte~tinal tetrazolium and 0.17 mg/ml 9l alkaline bromochloroindolyl l ! pho~phatase pho3phate in 20;~ lX AP 9.5 buffer (2).
, 4. poly ABAP Same a~ 3 Same a~ 3 21 o~ the invention (polymerized 22 according to the

23' ~oreg~ing procedure)

24 Enzyme reactions were terminated by waqhing the

25' filter ~trip~ in 10 mM Trici:Cl, 1 mM EDTA, pH 7.5, after 15 26i or 150 min. of incubation at room temperature.

271 The results o~ the four a~3ay3 are a3 follow3.
28li Comparing the peroxida~e complexes with pho~phataqe 29'l oomplexe~1 lt wa~ ~ound that the peroxlda~e reaction~ were 301i rapid, and the ~enqiitivltie~ of the two ~ample~ of peroxida~e Il * Trade Mark ,j 62-~L2373~9 j! i i l ll complex were quite different. The ABC complex could detect i 13 pg in a 15 min. reaction whil0 Detek II visualized 1.6 pg in the same amount of time. This i~ ~hown by Figure 5A, lane~ 1 and 2. This difference may be ~imply a function of Il the Qub~trate~ employed.
6 1' The phosphatase complexe~ had similar detection capabilities in a 15 min. reaction, that is 13 pg for ABAP
and 3 pg for poly ABAP. See Figure 5, laneq 3 and 4.
However, the phosphatase reactions continued to generate more ignal in the course of a 2.5 hr reaction while the i' peroxidase reactions only increased 2-fold at best in this extended reaction, see Figure 5B. In addition~ the 2.5 hr Detek II reaction generated heavy background staining o~ the nitrocellulo~e and a false positive reaction from the carrier protein spot No. 9 (Figure 5B, lane 2). The detection limits after 2.5 hr were 13 pg (ABC) Figure 5B lane 1, approximately 3 pg (Deteck II Figure 5B lane 2), 3-6 pg (ABAP; Figure 5A, lane 3) and 0.8 pg (poly ABAP; Figure 5B, lane 4).

I T~e specific efPect of enzyme polymerization on the 20l sensitivity of detection is shown by comparing th~ Figure 5 i results of ABAP and poly ABAP detection systems after either 15 or 150 min. These incubations clearly indicate that there 23!l 1 is an 8-to 16-fold increase in ~ignal for the polymeric !l enzyme complex of the invention (Figure~ 5A and B 9 lanes 3 25 ll and 4)

26 1I The Utilization of Avidin-Biotinylated Alkaline

27 j Phosphata~e Complexes in Immunocytochemical 'l Detection of_Human Chorionic Gonadotophin _ 28il !! A compari~on study was undertaken to determine the 29 !
j' usefulness of biotinylated polymer~ of alkaline phosphatase 30!1 Il in immunocytochemistry. The avidin-biotinylated polymer~ of !1 63 3~69 1, i .i ~
I' i 1 alkaline pho~hatase method (poly ABAP) was compared to the ¦ peroxida~e anti-peroxidaqe (PAP) technique of Sternberger, I¦ Sternberger et. al., J Histochem., Cytochem. 7 18, 3156 4 l (1970).
Ti3~ue ~ection~ from cancerous organ~ containing human chorionic gonadotropin were prepared on slide~ as 7,! follows:
Il Section~ were dewaxed in Xylene twice for 5 min.
il eachj then hydrated to distilled water through graded 10 ! alcohols. Section~ for peroxidase anti-peroxida~e ~PAP) ~ technique were treated with 1.5% H20 in P.B.S. for 30 12 l min. to block endogenou~ peroxida~e. Section~ ~or ABAP
13 technique were carried to ths next step without 14 l` treatment.
The section~ were treated with the following j ~uppre~or ~y~tem3:
17 ll For the PAP technique the sections were treated }8 with 10% Normal Swine Serum in 3% BSA in 0.05 M Tris.
9 ~! HCl buffer ph 7.5 for 20 min.
20,i For the polyABAP technique~ the seotions were 212l treated only with 3%BSA in 0.05M Tris.HCl buffer pH 7.5 I for 20 min. The ~ction~ were taken mounted on slides.
23 l¦ Primary antibody (Rabbit anti-HCG, Accurate Chem 24 1l : and Scientific Corp) wa~ diluted from 1:1000 to 25 ~ 1,000,000 in Tri~.HCl buffer pH 7.5 and applied to the 26 l ~lides both for 1 hour at room temp. and oYernight in a 27il ¦¦ wet chamber at room temperature. The ilide~ were wa~hed 28jl three timeqi with Tri~.HCl buffer pH 7.5.
29'1 I Secondary Serum wa~ then added.
For the PAP technique, a 1:20 dilution o~ Swine -64- i lZ31'~16~
. I , lli anti Rabbit antiserum (Accurate Chemical and Scientific 2i¦ Corp.) wa~ applied to the tis~ue ~ection for a half hour l at room temperature. For the poly ABAP technique, a 4l 1:400 dilution of biotinylated goat anti-rabbit 5l antiserum (Vector Laboratories, Inc.) diluted in 6 i Tris.HCl buffer pH 7.5 was applied for one half hour at 7,l room temp. In addition biotin labelled swine anti-8,¦ rabbit antiserum wa~ prepared and applied to the tissue l! sections in separate runs. Slides were then washed 10ll three times in Tris HCl. buffer for 5 min. each.
The Detection System was then added. For the PAP
12 !1 ~ystem, a 1:50 dilution of rabbit PAP was prepared in Tris HCl buYfer and applied to the slides for one hour at room temperature. For the polyABAP system, 20 ul. vf avidinDH was added to a borosilicate glass test tube ll containing 2.5 ml. of Tris HCl buffer pH 7.5 2.5 ul. of 17 l polymerized alkaline phosphatase was added and the 18 , mixture allowed 5 min to complex. The complex was then applied to the slide for one hour at room temperatureO
~I The slides were washed three times at room temp in Tris 21 HCl Buffer pH 7.5 The development system was then addedO For the PAP
¦ technique, slides were developed in diaminobenzidine 2sli! tetrahydrochloride and H202. For the polyABAP

26 1I technique, slides were developed in Napthol AS Phosphate jl and Fast Red TR Salt diluted in 0.05 M Tris HCl buffer 27 1l

28,l pH 9.5 in 0.1 M NaCl with 50mM MgC12 ~or 20 mln. at room

29~1 temp.

30~1 The ~lides were mounted and coverslipped and I examined by light mlcroseopy.

il 65-~ l l 3~6~3 1 ., !

il !
l l The re~ults are illustrated in Figure~ 6 and 7 as pictures of the slide~.
Figure 6 ~hows the reqult~ of the PAP technique at i 1:16,000 dilution.
5 , Figure 7 shows the re~ult~ of the polyABAP
Il technique at 1:160,000 dilution.
8 ~, It has been found that with overnight incubationq 1, and dependin~ upon the primary antibody, the polyABAP
Il technique of the invention is 4 to 20 times more sensitive ¦ than PAP. At short primary antibody incubations of one hour, the technique of the invention is 4 to 6 times more sen~itive, The increa~ed ~ensitivity noted in overnight li incubations is related to the ability of the polyABAP
14 ll ,~ technique to increase its ~en~itivity while the PAP method ~l remain~ at about the original titer, 16 i 271l 1 ~8,1 1 29~
30l~
i~ I

-6~-

Claims (52)

What is claimed is:

1. A method for visualizing the presence of an inorganic or organic target molecule in a biological material, which comprises:
combining said target with a visualization polymer comprising multiple units of a visualization monomer directly bonded together or linked by a coupling agent through chemical groups or backbone moieties of said units;
said visualization monomer having at least one visualization site and being selected from an enzyme, a tagged natural or synthetic polypeptide, a tagged polyol, a tagged polyolefin or a tagged carbohydrate;
said chemical group being a amine group, an oxidized l,2-diol group, a carboxy group, a mercaptan group, an hydroxy group or a carbon-hydrogen bond;
said backbone moiety being an amide bond, a carbon-carbon bond, a carbon-oxygen bond, or a carbon-hydrogen bond, and said chemical group or backbone moiety being located within said monomer at a position which is at least one atom away from the visualization site of said monomer.

2. A method according to claim 1 wherein the tag is selected from a fluorescent chemical group, a dye, a radioactive group, a photon emitter or an electron dense moiety.

3. A method according to claim 1 wherein said target is produced by or has an effect upon a biological organism.

4. A method according to claim 3 wherein said target is a protein, a lipid, a carbohydrate, a hormone, a steroid, a polynucleotide, a nucleic acid, a nucleoprotein, a nucleoside, a nucleotide, a purine or pyrimidine base, a drug, a sugar, a carcinogen, an antibiotic, a haptene an antigen, a heavy metal, an organometallic compound, a metal-protein complex or a toxic inorganic salt.

5. A method according to claim 2 wherein the coupling agent is derived from a reactive bi or multi homo or heterofunctional organic cross-linking reagent.

6. A method according to claim 5 wherein the cross linking reagent has formula I

wherein A, B and E are independently selected from hydrogen, a carboxylic acid group, an acid halide group, an activated ester, a mixed anhydride, an acyl imidazole, an N-(carbonyloxy)imide group, an iminoester, a primary amine, an aldehyde group, an alphahalomethylcarbonyl group, a hydrazine group, an acylhydrazide group, an azide group or an N-maleimide group, at least two of A, B and E are other than hydrogen; and R1 is an aliphatic group of at least two carbons or an aromatic group of at least six carbons.

7. A method according to claim 1 comprising combining said target with a detecting agent for said target, which agent carries said visualization polymer.

8. A method according to claim 7 wherein said detecting agent is an antibody, a lectin, a DNA repressor protein, avidin, streptavidin, a hormone, a stereospecfic receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein, or a complementary polynuoleotide sequence.

9. A method according to claim 7 wherein said detecting agent carries said polymer through an intermediate ligand binding complex comprising a first ligand covalently bonded to said agent, a second ligand covalently bonded to said visualization polymer and a ligand binding compound wherein said first and second ligands are complexed with said compound.

10. A method according to claim 9 wherein said compound is an antibody, lectin, avidin, streptavidin, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

11. A method according to claim 9 wherein the agent is an antibody, the compound is avidin or streptavidin, and the first and second ligands are independently selected from N-(omega acyl) amido [biotin or iminobiotin], said acyl group being of from 4 to 20 carbons; or an N-(omega-oligomer)amido [biotin or iminobiotin] wherein the oligomer is a polyol, a polyamide or polyvinyl group of 2 to 30 units in length.

12. A method according to claim 11 wherein a first target-specific antibody is used to detect said target and form a target/first antibody conjugate, a second antibody having said biotin label ligand and being general for said first antibody is incubated with said conjugate and said visualization polymer is complexed with said second antibody through said first and second ligands and said ligand binding compound.

13. A method according to claim 11 wherein the molecular ratio of visualization polymer to avidin or streptavidin is about 3 to 1.

14. A method according to claim 9 wherein the detecting agent is a lectin, the compound is avidin or streptavidin, and the first and second ligands are independently selected from N-(omega acyl) amido (biotin or iminobiotin), said acyl group being of 4 to 20 carbons; or an N-(omega-oligomer)amido [biotin or iminobiotin] wherein the oligomer is a polyol, a polyamide or a polyvinyl group of from 2 to 30 units.

15. A method according to claim 14 wherein the molecular ratio of visualization polymer to avidin or streptavidin is about 3 to 1.

16. A method according to claim 9 wherein said detecting agent is a complementary polynucleotide sequence.

17. A method according to claim 16 wherein said compound is an antibody, lectin, avidin, streptavidin, or a high affinity enzyme.

18. A method according to claim 16 wherein said compound is lectin and said first and second ligands are binding sugars.

19. A method according to claim 16 wherein said compound is avidin or streptavidin and said first and second ligands are independently selected from N-(omega acyl) amido (biotin or iminobiotin), said acyl group being of from 4 to 20 carbons; or an N-(omega-oligomer)amido [biotin or iminobiotin] wherein the oligomer is a polyol or polyamide or polyvinyl group of 2 to 30 units in length, or N-(alkenyl)amido (biotin or iminobiotin) having from 3 to 20 carbons in the alkenyl group.

20. A method according to claim 19 wherein the molecular ratio of visualization polymer to avidin or streptavidin is about 3 to 1.

21. A method according to claim 7 wherei- the visualization polymer is bonded to the detecting agent by a organic bivalent linking group.

22. A method according to claim 21 wherein the organic bivalent linking group is a derived from a bi-homo-or heterofunctional organic cross-linking reagent.

23. A method according to claim 21 wherein the detecting agent is an antibody, lectin, avidin, streptavidin, a DNA repressor protein, a hormone, a stereospecific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

24. A method according to claim 6 wherein the chemical groups are epsilon or primary amino groups which form amide bonds with the cross-linking reagent of formula I.

25. A method according to claim 24 wherein said monomer units are peroxidase, luciferase, glucose oxidase, galactoxidase, acid phosphatase or alkaline phosphatase.

26. A method according to claim 6 wherein the chemical group is a 1,2-diol which is oxidized to a dialdehyde group and the cross-linking reagent forms hydrazone, amide, or acylhydrazide or imine bonds with said dialdehyde group.

27. A method according to claim 6 wherein the chemical group is a mercaptan and the cross-linking reagent forms sulfide bonds using an alphahalomethylcarbonyl group or an N-maleimide group.

28. A visualization polymer comprising multiple units of a visualization monomer having at least one visualization site; said units being directly bonded together through chemical groups or backbone moieties of adjacent said units, or linked by a coupling agent covalently bonded to chemical groups or backbone moieties of adjacent said units;
said monomer being an enzyme, a tagged natural or synthetic polypeptide, a tagged polyol, a tagged polyolefin or a tagged carbohydrate; said chemical group being an amine group, an oxidized 1,2-diol group, a carboxy group, a mercaptan group, a hydroxy group or a carbon-hydrogen bond; said backbone moiety being an amide bond, a carbon-carbon bond, a carbon-oxygen bond or a carbon hydrogen bond; and said chemical group or backbone moiety being located at a position which is at least one atom away from the visualization site of said monomer.

29. A polymer according to claim 28 wherein said tag is selected from a fluorescent group, a dye, a radioactive group, a photon emitter or an electron dense group.

30. A polymer according to claim 29 wherein the coupling agent derived from a bi- or multi-(homo- or hetero-functional organic cross-linking reagent.

31. A polymer according to claim 30 wherein the cross-linking reagent has formula I
A B

E

wherein A, B and E are independently selected from hydrogen, a carboxylic acid group, an acid halide group, an activated ester, a mixed anhydride, an acyl imidazole, an N-(carbonyloxy)imide group, an iminoester, a primary amine, an aldehyde group, an alphahalomethylcarbonyl group, a hydrazine group, an acylhydrazide group, an azide group or an N-maleimide group; at least two of A, B and E are other than hydrogen; and R1 is an aliphatic group of at least two carbons or an aromatic group of at least six carbons.

32. A polymer according to claim 31 wherein the monomer is an enzyme selected from peroxidase, galactosidase, luciferinase, glucose oxidase, acid phosphatase or alkaline phosphatase.

33. A polymer according to claim 32 wherein the cross-linking reagent is an aliphatic-1, omega-diacyloxy disuccinimide having from 4 to 20 carbons in the aliphatic group.

34. A detection-visualization arrangement comprising a detecting agent carrying a visualization polymer as described according to claim 28, said agent being an antibody, a lectin, avidin, streptavidin, a DNA repressor protein, a hormone, a stereospecific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

.

35. A detection-visualization arrangement according to claim 34 wherein said detecting agent carries said polymer through an intermediate ligand binding complex comprising a first ligand covalently bonded to said agent, a ligand binding compound and a second ligand covalently bonded to said visualization polymer; said agent, compound and polymer forming a complex with said first and second ligands functioning as ligands to said compound.

36. An arrangement according to claim 34 wherein said agent is an antibody, a lectin or a complementary polynucleotide sequence, said compound is avidin or streptavidin, and said first and second ligands are independently selected from N-(omega acyl)amido(biotin or iminobiotin), said acyl group being of from about 2 to 20 carbons in length; or an N-(omega-oligomer)amido[biotin or imido biotin] wherein the oligomer is a polyol, a polyamide or a polyvinyl group of from about 2 to 30 units in length;
or N-(omega alkenyl)amido(biotin or iminobiotin), said alkenyl group being about 3 to 20 carbons in length.

37. A detection-visualization arrangement according to claim 34 wherein said polymer is bonded or complexed directly with said agent.

38. An arrangement according to claim 37 wherein said agent is covalently bonded to said polymer with a linking group derived from a bi- or multi-(homo or hetero) functional organic cross-linking reagent.

39. A visualization polymer complex comprising a visualization polymer according to claim 28 covalently bonded to a ligand, and a ligand binding compound associated with said ligand.

40. A complex according to claim 39 wherein the compound is a lectin, avidin, streptovidin, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

41. A complex according to claim 39 wherein the compound is a lectin, avidin or streptovidin.

42. A complex according to claim 41 wherein the compound is avidin or streptavidin and the ligand is an N-(omega acyl)amido(biotin or iminobiotin), said acvl group being of from about 2 to 20 carbons in length; or an N-(omega-oligomer)amido[biotin or iminobiotin]; said oligomer being a polyol, a polyamide or a poyvinyl group of from about 2 to 30 units in length.

43. A complex according to claim 41 wherein the compound in lectin and the ligand is a sugar bound to the polymer through a bi- or multi-(homo or hetero) functional organic cross-linking reagent.

44. A detection kit for analysis of target molecules in a biological material comprising metered quantities of a buffered, aqueous solution of a detection agent component and a buffered, aqueous solution of a visualization polymer complex component as described according to claim 39 and a standardized quantity of an aqueous mixture of said visualization polymer, said agent component and said polymer complex component being capable of associating when combined.

45. A kit according to claim 44 wherein said agent is an antibody, a lectin, avidin, streptavidin, a DNA
repressor protein, a hormone, a stereospecific receptor protein, a high affinity enzyme, a sequence specific polynucleotide binding protein or a complementary polynucleotide sequence.

46. A detection kit for analysis of target molecules in a biological material comprising a metered quantity of a detecting agent-visualization polymer arrangement as described according to claim 34.

47. A complex according to claim 42 wherein the ligand is N-(omega acyl)amido(biotin or iminobiotin).

48. A polymer according to claim 32 wherein the monomer is horseradish peroxidase or alkaline phosphatase.

49. A polymer according to claim 48 wherein the monomer units are linked by a coupling agent derived from disuccinimidyl suberate which bonds with epsilon amine groups.

50. A polymer according to claim 48 wherein the monomer is peroxidase and the units by a coupling agent derived from tri(lysyl)lysine which bonds with oxidized 1,2-diol groups.

51. A polymer according to claim 28 wherein said units are directly bonded together through oxidative cross-linking using an oxidative enzyme.

52. A polymer according to claim 51 wherein the oxidative enzyme is horseradish peroxidase.