CA2043089A1 - Biologically active reagents prepared from carboxy-containing polymer, analytical element and methods of use - Google Patents

Abstract

BIOLOGICALLY ACTIVE REAGENTS PREPARED FROM
CARBOXY-CONTAINING POLYMER, ANALYTICAL ELEMENT
AND METHODS OF USE
Abstract of the Disclosure Biologically active reagents are prepared from particles of copolymers having highly reactive carboxy or equivalent groups. The reagents are prepared by covalently attaching biologically active substances, for example antibodies, to the particles, directly or indirectly through highly reactive carboxy groups on the particle surface. These reagents are used to advantage in analytical elements, methods for the detection of specific binding ligands (such as immunological species) and immunoassays, and in purification methods as affinity chromatography reagents.

Description

29 ~3~89 BIOLO~.ICALLY ACTIVE REAGENTS PREPARED FROM
CARBOXY-CONTAINING POLYMER, ANALYTICAL ~LEM~NT
AND MET~ODS OF USE
Field of the Invention 5The present invention relates to biologically active reagents prepared using polymeric particles. It also relates to analytical elements containing such reagents, and to immunoassays and specific binding analytical methods using them.
Further, it relates to an analytical purification method using the reagents. Thi~ invention can be used for various clinical, diagnostic, medical and research purposes.
_ck~rQund of the Invention 15There i~ a continuing need in medical practice and research, and in analytical and diagnostic procedures for rapid and accurate determinations of chemical and biological substances which are present in various fluids, such as biological fluids. For example, the presence of drugs, narcotics, hormones, steroids, polypeptides, metabolites, toxins, viruses, microorganisms or nucleic acids in human or animal body fluids or tissues must be determined rapidly and accurately for effective research, diagnosis or treatment.
In approximately the last twenty years, a wide variety of analytical methods have been developed to detect the substances noted above.
Generally, the state of the art has advanced to such a degree that analytical and diagnostic methods ha~e become highly reliable and suitable for automation or for use with test kits which can be readily used in doctors' offices or at home. Most of such methods rely on what are known in the art as "specific binding" reactions in which an unknown substance to be detected (known as a "ligand~') reacts specifically _3_ 2 Q ~ 9 and preferentially with a corresponding "receptor"
molecule. Most well known specific binding reactions occur between immunoreactants, such as antibodies and antigens (foreign substances which produce immunological responses), but other specific binding reactions (such as between avidin and biotin and a sugar with a lectin~ are well known.
Methods in the art using specific binding reactions generally require that one or more or both of the reactants be immobilized on a solid substrate of some type, so that unreacted (and generally water-soluble) materials can then be separated from the water-insoluble reaction product (often called a "complex"). In addition, such immobilized reactants can be used in affinity chromatography to remove a desired biologically active material ~rom a mixture of such materials.
~ iologically active substances have thus been immobilized to advantage on particulate substrates such as polymeric particles, animal and human erythrocytes, bacte~ial cells and other solid materials known in the art. For example, carrier particles prepared from epoxy-group containing monomers are described in US-A-4,415,700. Where polymeric particles have been used as carrier substrates, biologically active substances have been attached through reactive groups on the particle surface, such groups pxovided either from the polymer composition or from linking moieties attached to the particles. US-A-4,401,765 describes a number of reactive groups on polymeric particles.
Several advances in the art in this regard are described in Canadian Application Serial No.
S48,335, Canadian Application Serial No. 567,821 and EP-A-0 308 235 (corresponding to Canadian Application No. 558,443). These applications describe various _4~ 3 ~ ~ ~
means for attaching biologically active substances to polymeric particles having various reactive surface groups, including surface carboxy groups, such as groups provided by acrylic and methacrylic acids.
Carboxylated latex particles have also been used to prepare diagnostic reagents, as noted in US-A-4,181,636. The described particles are prepared using a carboxyl-containing monomer such as acrylic acid, methacrylic acid, itaconic acid, aconitic acid, fumaric acid or maleic acid. Similar particles are described in US-A-3,857,931, US-A-4,138,383 and US-A-4,264,766.
Two commercially available monomers, 3-acrylamido-3-methylbutanoic acid and 2-acrylamido-2-hydroxyacetic acid, have been polymerized to form polymers. These monomers are generally water-soluble and are difficult to copolymerize with oleophilic monomers and are not readily polymerized to form monodisperse particles.
Another advance in the art relates to the use of specific compounds to attach biological materials to particulate substrates having reactive carboxy groups. Generally, water-soluble carbodiimides have been used, as described in the references noted above. More recently, however, carbamoylonium compounds have been used for this purpose with considerable advantages, as described in Canadian Application Serial No. 558,443. Dication ethers are also known to be useful, as described in Canadian Application Serial No. 612,926.
The modification of protein adsorption on polymeric surfaces has been a common goal for many workers trying to apply polymer technology to in vivo and i vitro uses in biotechnology. Undesirable protein adsorption has been a continual problem. For example, nonspecific adsorption is a major concern in -5~ $
the use o~ polymers for affinity chromatography for the purification of proteins.
The modification of polymer surfaces has taken many forms, including physical coatings, graft copolymerization, chemical treatments and plasma gas discharge treatment. The hyclrophilic nature of the polymer surface has been the subject of considerable debate and research because an increase in hydrophilicity reduces adsorption of some proteins, but no~ others. As noted in the art cited above, the use of reactive side chains has also received considerable attention in the art.
There is a need in the art to find new biological reagents which show improvement over the standard reagents prepared from standard carboxy-containing polymers. Such reagents would be especially useful having attached biological materials for use in research and various analytical and diagnostic procedures.
Summarv of the Invention The problems noted with known reagents are overcome with a biologically active reagent comprising:
(I~ a water-insoluble particle composed of, at least on its æurface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to the copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive earboxy group, or salt thereof, and represented by the -6~ 3 ~ ~ ~
structure:

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an a~kali meta~ ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen ~nd sulfur atoms in the linking chain, and ~ c> from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b~ above, and (II) a biologically active substance covalently attached to the particle through the reactive carboxy group or salt thereof.
This invention also provides an analytical element comprising a substrate having one or more reaction zones therein, and containing in at least one of the zones, a biologically active reagent as described above.
Moreover, a method for the determination of a specific binding ligand comprises:
A. forming a water-insoluble specific binding complex of a specific binding ligand of interest, or a receptor therefor, with a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to the copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated _7_ 2 ~ 9 polymerizable monomers having a reactive carboxy ~roup, or salt thereof, and represented by the structure:

CH2=C--L--C--O--M
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking ~roup having from ~ to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a biologically active substance covalently attached to the particle through the reactive carboxy group or salt thereof, said substance being specifically reactive with either the ligand or a receptor therefor, and B. detecting the presence of the complex as an indication of the presence or amount of the ligand in the specimen.
This invention also provides an assay for the determination of a specific binding ligand comprising:
detecting the presence or amount of a water-insoluble specific binding comple~ formed between a ligand oX interest and a receptor therefor, the receptor provided as a component of a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer as described above, and 8~
~ II) the receptor for the ligand being covalently attached to the particle through the reactive carboxy group or salt thereof.
Still further, an immunoassay employing antibodies or antigens for detecting the presence or amount o~ a ligand in a specimen comprises addition of an immunoreactant which i9 specifically reactive with the ligand or with a receptor therefor, the immunoreactant being a component of a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer as described above, and tII) the immunoreactant being covalently attached to the particle through the reactive carboxy group or salt thereof.
An analytical separation method of this invention comprises:
A. passing a specimen containing a mixture of biologica~ly active substances over an affinity chromatography reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer as described above, and (II) a specific binding substance covalently attached to the particle through the reactive carboxy group, the specific binding substance being specific to one or more predetermined biologically active substances in the specimen mixture of biologically active substances to form a complex of the reagent with the predetermined substances, and B. collecting either the one or more complexed predetermined substances or one or more substances remaining in the eluent.
The present invention provides reagents which are useful in a variety of analytical, 9_ 2~ 3~
diagnostic and purification methods. These reagents are improvements over known reagents prepared using standard carboxy-containing polymers.
The advantages of the present invention are provided ~y the use of certain copolymers having a carboxy group which is extended from the copolymer surface by a sufficient length to allow improved results in the attachment of biologically active substances and their subsequent use. Thus, in the structure noted above, the organic group identified as "L~ is critically from 8 to 50 carbon, nitrogen, oxygen and sulfur atoms in length.
The extended hydrophilic carboxy group on the monomers used to make the copolymers provide certain advantages over monomers having shorter carboxy groups which are known in the art. During emulsion polymerization, the improved monomers have less tendency to polymerize in the aqueous phase as solution (or water-soluble) polymers. Thus, the improved monomers are more easily and more completely incorporated into water-insoluble latex particles, and thereby facilitate attachment of proteins or other biological compoundF.. Latices prepared from acrylic acid contain unwanted solub~e polymer in the aqueous phase, which for some uses, must be removed at considerable expense. The improved monomers described herein produce less water-soluble polymer.
Further, the reactivity ratios of the preferred monomers used in the practice of this invention, that is those having aromatic groups as part of L (such as styrene derivatives), are more favorable than known carboxy-containing monomers for polymerization with aromatic comonomers, such as styrene and styrene derivatives. Moreover, the extended linking group enables the carboxy ~roups to be more easily activated by carbodiimides or other -lo~ 5~ 9 activation agents when biological compounds are attached to the particles.
etailed Description of the Invention Many of the copolymers useful in the preparation of the reagents of this invention and methods of preparing same are described in detail in Canadian Patent Application (corresponding ~to USSN 539,768 and USSN 654,112). The following discussion is provided as a ~'ummary of the copolymers useful in this invention.
The copolymers have as an essential component recurring units derived from one or more ethlenically unsaturated polymerizable monomers having the following structure:

CH2=C-L-C-0-M
wherein R is hydrogen~ halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 carbon, oxygen, nitrogen or sulfur atoms in the linking chain. A mixture of monomers can be used if desired, although preferably only one such monomer is used to prepare each copolymer.
More specifically, in the structure noted above, R is hydrogen, halo (such as chloro or bromo) or alkyl of 1 to 3 carbon atoms (such as methyl, ethyl, isopropyl and n-propyl~. More preferably, R
is hydrogen or methyl.
Also, M is hydrogen, an alkali metal ion (such as lithium, sodium and potassium) or an ammonium ion (such as ammonium, tetramethylammonium and tetraethylammonium). Preferably, M is hydrogen or an alka:Li metal ion, and more preferabLy, it is hydrogen or sodium.

L is an organic linking group which has from 8 to 50 of a combination o~ carbon, nitrogen, oxygen or sulfur atoms in the chain. The linkage comprises two or more divalent hydroca:rbon groups such as alkylene, arylene, alkylenearylene, arylenealkylene and similar groups which are connected or terminated with the noted heteroatoms or with heteroatom-containing groups such as carbonyl, sulfonyl, imino and otheræ known in the art. Such hydrocarbon groups can have .from 1 (such as methylene) up to 1~ carbon atoms, and can be branched, linear or cyclical, substituted or unsubstituted with one or more alkyl groups (preferably of f rom 1 to 12 carbon atoms, such as methyl, ethyl, isopropyl, hexyl and octyl), alkoxy (preferably from 1 to 12 carbon atoms, such as methoxy, ethoxy, propoxy, t-butoxy and octyloxy), cycloalkyl (preferably from 4 to.6 carbon atoms, such as cyclobutyl, cyclohexyl and cyclopentyl), aryl (preferably from 6 to 12 carbon atoms, such as phenyl, tolyl, xylyl, naphthyl, 4-methoxyphcnyl and chlorophenyl). Such groups ase not difficult to design or synthesize for one skilled in synthetic chemistry.
Preferably, L comprises two or more alkylene or arylenealkylene groups which are connected or terminated with an oxy, thio, imino (-NRl-), carbonyloxy (-COO-), carbonylimino (-CONRl-), ureylene (-NRlCONRl-) or sulfonylimino ~-S02NRl-) group, wherein each Rl in the noted groups is independently hydrogen, alkyl having 1 to 10 carbon atoms (such as methyl, ethyl, isopropyl, n-butyl, hexyl, benzyl and 2,4-dimethylpentyl), cycloalkyl having 4 to 10 carbon atoms in the backbone (such as cyclopentyl, cyclohexyl and 1,3-dimethylcyclohexyl) or aryl having 6 to 14 carbon -12- 2 ~ft3 atoms in the backbone (such as phenyl, xylyl, p-chlorophenyl, naphthyl and anthryl).
Representative L groups include, but are not limited to:
~-phenylenemethyleneoxycarbonyltrimethylene, carbonyloxyethyleneoxycarbonyltrimethylene, carbonyloxyethyleneureylenepentamethylene, carbonylpenta(oxyethylene)o~ycarbonyltrimethylene, carbonyldeca(oxyethylene)oxycarbonyltrimethylene, ~-phenylenemethylenethioethyLeneoxycarbonyltri-methylene, carbonyloxyethyleneiminocarbonyl-trimethylene, carbonyloxytetramethyleneoxycarbonyl-tetramethylene, ~-phenylenemethyleneiminocarbonyl-trimethylene, ~-phenylenemethyleneiminocarbonyl-trimethylene, ~-phenylene(methyl)iminoethyleneo~y-carbony~trimethylene, ~-phenylenemethylenethio-ethylene, P-phenylenemethylenethioethyleneimino-carbonylmethyleneoxymethylene, ~-phenylenemethylene-thioethyleneiminocarbonylmethylenethiomethylene, p-phenylenemethylenethioethyleneiminocarbonyltri-methylene, phenylenemethylenethio-l-carboxyethylene, phenylenemethylenethiophenylene, phenylenemethylene-thioethyleneoxyethylenethiomethyleneoxycarbonyl-ethylene, phenylenemethyleneoxyphenylenemethylene-~5 thioethylene, phenylenemethylenethioethyleneoxy-ethylenethioethyleneoxycarbonylethylene, phenylene-methyleneoxyphenylenemethylenethiophenylenemethylene-thiotrimethylene and phenylenemethylenethioethylene-oxyethylenethioethyleneoxycarbonylphenylene.
Representative monomers described by the structure identified above include, but are not limited to: mono-m & ~-vinylbenzyl glutarate, mono-~-vinylbenzyl glutarate, mono-~-methacryloyloxyethyl glutarate, 2-(4-carboxybutyramido)ethyl methacrylate, 2-[N'-(5-carboxypentyl)ureido]ethyl methacrylate, mono-methacryloylpenta(oxyethylene) ~lutarate, mono--13- ~4~
~4-acryloyloxybutyl> glutarate, 4-(4-carboxybutyr-amido)styrene, mono-methacryloyldeca(oxyethylene~
glutarate, mono-2-(~-vinylbenzylthio~ethyl glutarate, mono-2-(_- & ~-vinylbenzylthio)ethyl glutarate, 4-(4-carboxybutyramidomethyl)styrene, mono-2-[N-methyl-~-(4-vinylbenzyl)amino]ethyl glutarate, 3-(p-vinylbenzylthio)propionic acid, 4-[2-(4-carboxybutyramido)ethylthiomethyl]styrene, 4-[2-(carboxymethoxyacetamido)ethylthiomethyl]styrene, 10 4-~2-(carboxymethylthioacetarnido)ethylthiomethyl]-styrene, mono-2-(4-vinylbenzylthio)ethyl succinate, 4-[2-(carboxymethoxyacetoxy)ethylthiomethyl]styrene, mono-4-vinylbenzyl succinate, 2-(4-vinylbenzylthio)-succinic acid, 2-(4-vinylbenzylthio)benzoic acid, mono-2-[2-~4-vinylbenzylthio)ethoxy~ethylthiomethyl malonate, mono-methacryloylpenta(oxyethylene>
phthalate, mono-methacryloyldeca(oxyethylene) phthalate, mono-2-{2-[2-(4-vinylbenzylthio)-ethoxy]ethylthio}ethyl succinate, mono-2-{2-[2-(4-vinylbenzylthio)ethoxy]ethylthio}ethyl phthalate, 3-[4-(4-vinylbenzyloxy)benzylthio]-propionic acid and 4~{4-[4-(4-vinylbenzyl-oxy)benzylthio]benzylthio}butyric acid.
The most preferred monomer is 3-(~-vinyl-benzylthio)propioniC acid.
The monomers described above are generallycopolymerized with one or more additional ethylenically unsaturated polymerizable monomers.
The oleophilic monomers identified above as (a) monomers are useful for providing hydrophobicity or water-insoluble properties to the resulting copolymer. A mixture of such monomers can be used if desired. Such monomers include, but are not limited to, vinyl aromatics (for example, styrene and styrene derivatives such as 4-vinyltoluene, ~-methyl-styrene, ~,5-dimethylstyrene, 4-t-butylstyrene and -14- 2~ ~9~
2-chlorostyrene), acrylic and methacrylic acid esters and amides (~or example, methyl acrylate, methyl methacrylate, _-butyl acry~ate, 2-ethylhexyl meth-acrylate, benzyl acrylate and N-phenylacrylamide), butadiene, acrylonitrile, vinyl acetate, vinylbenzyl acetate, vinyl bromide, vinylidene chloride and crosslinkable monomers having two or more polymer izable groups. Useful crosslinkable monomers include, but are not limited to, divinylbenzene, allyl acrylate and di- and triacrylates and methacrylates (such as 2,2-dimethyl-1,3-propylene diacrylate, 1,4-cyclohexylenedimethylene dimethacrylate, ethylene diacrylate, ethylene dimethacrylate, propylene diacrylate, propylene dimethacrylate, ethylidyne trimethacrylate) and others readily apparent to one skilled in polymer chemistry.
In addition, ethylenically unsaturated polymerizable monomers (c) other than those described above for monomers (a) or (b) can be copolymerized to provide desirable properties. For example, such monomers include anionic monomers containing sulfonic acid groups or salts thereof, including 2-acryl-amido-2-methylpropane sulfonic acid, 3-methacryloyl-oxypropane-l-sulfonic acid, ~-styrene sulfonic acid and salts thereof, and others readily apparent to one skilled in the art. Also included in the (c) group of monomers are nonionic hydrophilic monomers such as acrylamide, methacrylamide, N-isopropylacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, N-vinyl-2-pyrrolidone and others readily apparent to one skilled in the art. In addition, monomers having active methylene groups, such as 2-acetoacetoxyethyl methacrylate, and cationic monomers, such as N,N,N-trimethyl-N-vinylbenzylammonium chloride and 3-hydroxyethyl-1-vinylimidazolium chloride could be -15- 2~ ~3~g~
used, as well as many others too numerous to mention here. A skilled polymer chemist would be able to readily fashion useful polymers from hundreds of available or producible monomers using the teaching presented herein.
Generally, the copolymers of this invention are composed of recurring un;ts derived from about 60 to about 99.8 mole % of (a), from about 0.2 to about 40 mole % of (b), and from 0 to about 15 mole % of (c). Preferred copolymers are prepared from about 85 to about 99.5 mole % of (a), from about 0.5 to about 15 mole % of (b), and from 0 to about 10 mole % of ( c ) .
The copolymers of this invention are prepared using standard emulsion or suspension polymerization techniques, as described for example by Sorenson et al in Preparative Methods of Pol~mer nce, 2nd Ed. (1968~,-Wiley and Sons, New York, and Stevens, ~lYmer Chemistry. An Introduction, Addison Wesley Publishing Co., London, 1975, and certain preferred conditions are discussed in copending Canadian Patent Application (identified above).
The copolymers described herein are used in particulate form in order to prepare the reagents of this invention. The average particle size can vary greatly depending upon reagent use. Generally, it is ~rom about 0.01 to about 20~m, and preferably from about 0.1 to about lO~m.
The reagents of this invention have one or more biologically active substances covalently attached to the polymeric particles through the reac~ive carboxy groups on the outer surface of the particles. As used herein, the term "biologically active substance" is meant to include any organic compound which is found in a living organism or which -16~
is useful in the diagnosis, treatment or genetic engineering of cellular material or living organisms, and which has a capacity for interaction with another biological or chemical material. Such substances may or may not be naturally occu:rring in biological fluids. Such materials must be capable of attachment to the particles through the reactive ~arboxy groups using an appropriate activation agent, as described below. Thus, generally, this means that the biologically active substance has an available amino or sulfhydryl group for reaction.
Depending upon the intended use o~ the reagent, the biologically active substances can be from a wide variety of naturally occurring or synthetically prepared materials, including, but not limited to amines, enzymes, amino acids, peptides, polypeptides, proteins (including antibodies, C-reactive protein and avidin and its derivatives), lipoproteins, glycoproteins, hormones, drugs (for example digoxin, phenytoin, phenobarbital, thyroxine, triiodothyronine, gentamicifi~ carbamazepine and theophylline), steroids, vitamins, polysaccharides, glycolipids, a~kaloids, microorganisms, viruses, protozoa, fungi, parasites, rickettsia, molds, blood ~5 components, tissue and organ components, pharmaceuticals, haptens, lectins, toxins, nucleic acids (including oligonucleotides, either single- and double-stranded), antigenic materials (including proteins and carbohydrates), biotin or derivatives thereof, and components of any of the materials just listed, and others known to one skilled in the art.
Particularly useful reagents of this invention are those in which the biologically active substance is a receptor molecule specific to a ligand of interest. Thus, a specific binding reaction involving the reagent can be used for various methods (described in more detai~ below). Examples of ligand-receptor complexes (that is, reaction o~ the ligand and receptor) include, but are not limited to antibody-antigen, antibody-hapten, avidin-biotin, sugar-lectin, gelatin-fibronectin and Protein A-IgG
complexes. For purposes of this invention, complementary nucleic acids (that is, a hybridized product of complementary strands) are also considered specific binding materials. Such complementary nucleic acids (including oligonucleotides having at least 2 bases) need not be complementary at every base pair, nor must there be a matching base at every position in the nucleic acid sequence That is, one of the strands can be longer than the other, or one strand can have a plurality of oligonucleo~ides complementary thereto at difference sequences.
Most useful biologically active substances are what are known in the art as immunoreactive species which include: (1) any substance which, when presented to an immunocompetent host, will result in the production of a specific antibody capable of binding with that substance, or (2) the antibody so produced, which compound participates in an immunological reaction. Thus, the immunological species can be an antigenic material or an antibody (including anti-antibodies). Both monoclonal and polyclonal antibodies are useful, and they can be whole molecules or various fragments thereof, as long as they have at least one reactive site for reaction with the reactive carboxy groups on the particles.
Pàrticularly useful biologically active substances include antibodies directed to Streptococcus A, a microorganism associated with periodontal disease, carbamazepine, thyroxine, human chorionic gonadotropin, phenobarbital, phenytoin, digoxin or a C-reactive protein.

2 ~

In certain embodiments, the immunological species is an enzyme which has a reactive group for attachment. Representative enzymes include, but are not limited to, horseradish peroxidase, glucose oxidase, urease, ~-galactosiciase, aspartate aminotransaminase, alanine aminotransaminase1 lactate dehydrogenase, creatine phosphokinase, ~-glutamyl transferase, alkaline phosphatase, acid phosphatase and prostatic acid phosphatase.
ln other embodiments, such as for competitive binding assays for determination of drugs or pregnancy, the biologically active substance is an antibody directed to human chorionic gonadotropin, phenobarbital, phenytoin or digoxin.
If desired, the biologically active substance can be modified or chemically altered to provide reactive groups for attaching, including providing a linking moiety for attachment. There is considerable technology known in the art for such chemical modification or the use of linking moieties, including teaching in such references as US-A-4,914,210, and W0-A~89/2932, both directed to modification of oligonucleotides, US-A-4,719,182, Erlanger et al, J~iol.Çhem., 234, 1090 (1959~, Wiston et al, Biochim.Biophvs.Acta, 612, pp.40-49 (1980) and Borzini et al, J.Immunol.Methods, 44, pp.
323-332 (1981).
The biologically active substances are attached to particles of the copolymers described herein using activating agents. These activating agents are compounds capable of converting the carboxy groups into an intermediate (such as an ester) which is reactive with the available amine or sulfhydryl groups of the substance.
Useful activating a~ents include, but are not limited to N-ethyl-5-phenylisoxazolium--19- 2 ~
3~-sulfonate, chloroformate, the well known water-soluble carbodiimides, as described for example in US-A-4,181,636, dication ethers, carbamoylonium compounds and others readily apparent to one skilled in the art.
Particularly useful water-soluble carbo-diimides include, but are not limited to, l-cyclo-hexyl-3-[2-morpholinyl-(4)-ethyl]-carbodiimide metho-~-toluenesulfonate and l-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochlorlde.
Other useful activating agents are dication ethers such as those described in copending Canadian Application No. 612,926. Included among such compounds are bis(tetramethylformamidinium) ether ditriflate and bis(l-methyl-2-pyridinium) ether ditriflate.
Preferred activating agents are the carbamoylonium compounds described in considerable detail in EP-A-O 308 235. Useful carbamoylonium compounds include, but are not limited to, 1-(4-morpholinocarbonyl)-4-(2-sulfoethyl)- pyridinium hydroxide, inner salt, l-(l-pyrroli-dinylcarbonyl)pyridinium chloride and l-(4-morpho-linocarbonyl)pyridinium chloride. The most preferred compound for making most reagents of this invention is l-(l-pyrrolidinylcarbonyl)-pyridinium chloride.
The procedure for attaching a biolo~ically active substance to polymeric particles to prepare the reagents of this invention is generally as follows.
The general procedurè for preparing the reagent of this invention occurs in two steps. T~.e first step involves contacting an aqueous suspension (or latex) of the polymeric particles with an activating agent to produce reactive intermediates (for example, esters or mixed anhydrides> attached to -20~
the particles in place of the original carboxy groups. This step is carried out at a suitable pH
using acids or buffers that are appropriate. For the carbamoylonium compounds and dication ethers, the pH
is generally less than about 6 (preferably from about 3.5 to about 6). For carbodiimides, the pH i6 generally from about 4.5 to about 7. The amount of activating agent used in the pxeparation is generally at least about a two-old excess over the stoichiometric concentIation of total carboxyl groups in the polymeric particles, but the optimum amounts are readily determined by xoutine experimentation using the teaching provided herein and known in the art. For example, for the carbamoylonium compounds of EP-A-0 308 235, the molar ratio of the compound to carboxy groups on the particles is from about 1:1 to about 200:1, and preferably from about 100:1 to about 10:1. For preferred carbamoylonium activating agents, such as l-~l-pyrrolidinylcarbonyl)pyridinium chloride, the molar ratio of compound to carboxyl ~roups is from about 1:1 to about 200:1, and preferably from about 2:1 to about 100:1. For dication ethers, this ratio is generally from about 10:1 to about 1000:1, and preferably from about 50:1 to about 400:1.
In the reaction mixture, the % solids of particles is generally from about 0.01 to about 10%, and preferably from about 0.1 to about 5%, in preparing the reagent. The amount of biologically zctive substance is generally designated by a weight ratio of substance to copolymer of from about 0.0005:1 to about 0.5:1, and preferably from about 0.005:1 to about 0.1:1. ~owever, it should be understood that not all of the substance may become covalently bound to the particles. In fact, a minor amount may be adsorbed, and some may not be bound at 2~f~13~3~

all. One skilled in the art could readily perform tests to determine the amount of substance bound to the particles.
Mixing of the biologically active substance and particles is carried out at a temperature of from about 20 to about 37C for f:rom about 2 to about 30 houre. The length of time will vary with the temperature, activating agen~, biologically active substance and the desired coverage. Any sui~able buffer can be used, but 2-(N-morpholino)ethane sulfonic acid is preferred.
The details of representative procedures for making ~arious reagents are shown in the Examples below.
It is desired that the biologically active substance be present in the reagent in an amount of from about 0.0025 to about 30%, and preferably from about 0.005 to about 10%, by weight o~ the polymer particles. As noted abovej not all of the substance mixed with the particles may become bound. Hence, usually an excess of substance is mixed with the particles than actually becomes covalently bound.
In one embodiment of this invention, nucleic acid reagents for hybridization or other assays using water-insolubilized nucleic acids can be prepared using carboxylated polymeric particles, including but not limited to the specific carboxylated polymeric particles described above. Thus, the reagents can be prepared from particles composed of a polymer represented by the structure:
( A ~x ( B ~ OO-x wherein A represents recurring units derived from one or more ethylenically unsaturated polymerizable mono-mers containing carboxyl groups or salts or precur-sors of such groups, and B represents recurring units ~ 2 ~

derived from one or more ethylenically unsaturated polymerizable monomers.
Monomers from which A can be derived include, but are not limited to, acrylic and methacrylic acids, itaconic acid, aconitic acid, fumaric acid, maleic acid, ~--carboxyethyl acrylate, ~-carboxyethyl methacrylate, _ & p-carboxymethyl-styrene, methacrylamidohexanoic acid and N-(2-carboxy-l,l-dimethylethyl)acrylamide or a salt or anhydride precursor thereof. Monomers from which B
can be derived include, but are not limited to, styrene and styrene derivatives (for example vinyltoluene, 4-t-butylstyrene, divinylbenzene and 2-chloromethylstyrene), acrylic and methacrylic acid esters (for example, methyl acrylate, ethyl meth-acylate, n-butyl acrylate, 2-ethylhexyl methacrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, methacrylamide, ethylene dimethacrylate and 2-hydroxyethyl acrylate), sodium 2-acrylamido-2-methylpropanesulfonate, sodium 3-acryloyloxy-propanesulfonate, sodium ~-styrenesulfonate, or acrylonitrile. Preferably, B is deri~ed from styrene or a styrene derivative, or an acrylic or methacrylic acid ester.
For both the A and B monomers, it is important that the specific monomers used and their proportions be chosen so as to render the particles water-insoluble.
In the structure identi~ied above, x is from about 0.1 to about 70, and preferably from about 1 to about 20, mole percent.
The preferred monomers from which A is derived are those represented by the structure:

CH2=C-L-C-0-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, as further defined above.
The nucleic acid reagents are advantageously prepared similar to the othe:r reagents described above using an activating agent, but more particularly, the polymeric particles having an average particle size of from about 0.01 to about 20~m are present in the suspension in an amount of at least about 5% solids, and pre~erably from about 8% to about 25% solids. The advantage of this feature is that it produces a reagent that gives a much higher signal in an assay for cytomegaloviral DN~. The specific details of the preparation of these reagents is described in Example 15 below.
More generally, however, the method comprises:
A. contacting (l) an aqueous suspension of carboxylated polymeric particles having an average particle size of from about 0.01 to about 20~m, the particles being present therein at at least about 5%
solids with (2) an activating agent to produce reactive intermediate polymer particles having intermediate reactive groups, and B. contacting the reactive intermediate polymer particles produced in step A with an oligonucleotide having a reactive amine or sulfhydryl group which reacts with the intermediate reactive groups to form a covalent linkage between the particles and the oligonucleotide.
Where the oligonucleotide does not have the requisite reactive amine or sulfhydryl groups, they can be added using known procedures and reactants as described for example in US-A-4,914,210.

In the analytical or diagnostic methods of this invention, the reagents can be used to detect any specific binding ligand for which there is a receptor molecule. The biologically active substance in a reagent of this invention can be ~peci~ically reactive with either the ligand or its receptor.
Ligand detection can be carried out in solution or dry form (described below) using test specimens of aqueous ~luids (such as biological ~luids), or solutions of tissue or cellular materials, and can be quantitative, qualitative or both. In particular, the invention can be used to assay biological fluids of animals, humans or plants, but preferably Pluids of humans including whole blood, sera, plasma, lymph, bile, urine, spinal $1uid, sputum, lacrimal fluid, perspiration, swab specimens, tissue cultures, stool secretions, cellular fluids, vaginal secretions and semen. It is a~so possible to assay fluid preparations of human or animal tissue such as skeletal muscle, heart, kidney, lungs, brains, bone marrow or skin.
The ligand can be a drug, hapten, hormone, an antigenic material (lipopolysaccharide or protein) or antibody which has one or more sites for complexation with one or more of the same or different receptor molecules. In immunoassays of this invention, the ligand can be a drug ~such as digoxin, phenytoin and carbamazepine), a hormone (such as thyroid stimulating hormone, human chorionic gonadotropin, leutinizing hormone and thyroxine), retroviral component or an antibody to the retrovirus (such as an HIV-I component or its antibody), bacterial infectious agents or components thereof or antibodie~ thereto (such as Streptococcus A antigen, Chlamydial or Gonococcal antigen or antibody), viruses or components thereof (such as hepatitis, -25~
cytomegalovirus or herpes antigen) or antibodies thereto, cancer-producing agents, or C-reactive protein. The ligand can also be biotin or a derivative thereof, and the receptor is avidin or a derivative thereof.
In other embodiments, the ligand can be a nucleic acid (usually in single-stranded form), the amount or presence of which is detected using a complementary single-stranded nucleic acid as the receptor molecule. There are many various assay formats for nucleic acid detection, all of which are readily apparent to one skilled in the art.
Detection o HIV-I DMA, ~-globin DNA or cytomegalovirus DNA is of particular interest in the practice of this invention.
In general, a method for the determination of a specific binding ligand comprises:
A. forming a water-insoluble specific binding complex of a specific binding ligand of interest, or a receptor thereof, with a reagent comprising:
(I~ a water-insoluble, nonporous particle as described above, and (II) a biologically active substance covalently attached to the particle through the reactive groups, the substance being specifically reactive with either the ligand or a receptor therefor, and B. detecting the presence of the complex as an indication of the presence or absence of the ligand in the specimen.
In one embodiment~ the reagent can be used in competitive binding assays for determination of a specific binding ligand. In general, such an assay comprises:
A. contacting a specimen suspected of containing a water-soluble specific binding ligand -26~
with a water-soluble receptor therefor, and with a reagent as described above, to form a specific binding complex (a) between the receptor and the ligand, and specific binding S complex (b~ between the receptor and the water-insoluble reagent, and B. after separating complexes ~a) and (b~, detecting the presence of eit:her complex as an indication of the presence or amount of the ligand in the specimen.
Such competitive binding assays can be caIried out in solution. A solution assay is one in which the reagents are used in a suspension of reagent and test specimen suspected of containing the ligand of intere~t. EitheI bound (that is, complexed) or unbound ~that is, uncomplexed) materials can be determined in the assay. Physical separation of bound and unbound materials, if desired, can be carried out using any suitable separation technique. In using analytical elements (described below), either vertical or horizontal separation can be used. Bound ligand can be determined using light scattering, turbidimetric, radiometric or spectrophotometric techniques as are known in the art.
In a competitive binding assay, the reagent is generally present in a concentration which depends upon the amount of immunological species (that is, receptor) on the polymeric particles and the ligand of interest. A ligand analog (ligand which is detectably labeled) is also used so there is a competition between ligand and ligand analog for a known amount of receptor available for reaction. The assay is generally carried out by physically contacting and mixing the reagent, ligand analog and test specimen in a suitable container æo that -27- ~ 9 complexation occurs. Incubation may be used to promote complexation and any chemical or biological reactions (such as dye formation) needed for detection of the complexes.
More particularly, the ligand is an immunological species and the reaction of ligand and receptor therefor forms an immunological complex which is detectable once water~soluble (uncomplexed) materials are removed from the complex (for example, by filtration or centrifugation) to indicate the presence or absence of the species in the specimen.
The methods o~ this invention can also be carried out using dry analytical elements. The simplest element can be composed of an absorbent, fluid permeable substrate, for example, a thin sheet of a self-supporting absorbent or bihulous material such as a filter paper or paper strip. This substrate has one or more reaction zones for chemical, biological or specific binding reactions to occur therein. The reagent of this invention is present in at least one of these zones. Other - optional zones can include other reagents, such as dyes, dye-providing compounds, scavengers, antioxidants, enzyme substrates or buffers and other materials readily apparent to one skilled in the art. Such elements are known in the art as test strips, analytical elements, slides or dip sticks.
Absorbent materials useful in preparing the elements can include cellulosic materials (such as porous papers), porous polymeric films, mats of glass fibers, woven or nonwoven fabrics and other materials known to one skilled in the art. Preferred substrates are porous spreading layers as described, for example, in US-A-3,992,15~, US-A 4,258,001, US-A-4,292,272 and US-A-4,430,436.

-2~- ~? ~
Preferred elements can include one or more superposed fluid-permeable layers, all of which are superposed on a nonporous, fluid impermeable support (which can be transparent or not) composed of a suitable polymeric, cellulosic or metallic material.
The layers ean be used for various purposes, such as for reaction zones, subbing zones, reagent zones, barrier zones, radiation-blocking zones and other uses well known in the art. Where desired, reagents and buffers can move among the layers ~or the desired reactions to carry out the assay and provide a detectable product and separation of bound and unbound materials. Other components of analytical layers are described, for example, in US-A-4,042,335, US-A-4,132,528, US-A-4,144,306, US-A-4,670,381 and ~P-A-0 253 581.
While it is preferred that the reagent of this invention be incorporated into an element for use, this is not critical because the reagent can be added to the elemen~ at the time of the assay along with the test specimen. Preferably, however, the ligand analog and reagent o~ this invention (containing the appropriate receptor) are located within the element in different zones so they will not complex prematurely.
In one preferred embodiment of this invention, an analytical element comprises a nonporous support, having imposed thereon, in order and in fluid contact, a reagent layer containing one or more reagents for providing a detectable signal in the assay, a water-soluble layer containing a detectably labeled analog of a ligand of interest, and a porous spreading layer containing the reagent of this invention composed of a receptor (for example, an antibody) for the ligand of interest.

PIeferably, the ligand analog is labeled with a enyzme, such as one described below, the ligand is an antigenic material, hormone, hapten or drug, and the receptor is the corresponding antibody. Such elements are particularly useful for the determination of carbamazepine, thyroxine, phenobarbital, phenytoin or digoxin. Most preferably, they are useful for the determination of phenobarbital, phenytoin or digoxin.
A ~ariety of different elements, depending upon the method of assay, can be prepared according to this invention. They can be configured in a variety of forms, including elongated tapes of any desired width, sheets, slides or chips.
The solution or dry assay of this invention can be manual or automated. In general, in the use of dry elements, analyte determination is made by taking the element from a supply roll, chip packet or other source and physically contacting it with a sample of test specimen so the specimen and reagents within the element become mixed in one or more test zones. Such con~act can be accomplished in any suitable manner, for example, by dipping or immersing the element into the sample or, preferably, by 2~ applying a drop of the specimen to the element with a suitable dispensing means. Wash fluids can also be used in the assay, for example as described in US-A-4,517,288.
Assay results are generally determined by observing detectable spectrophotometric changes in the element either visually or with suitable detection equipment.
Another embodiment of this invention is what is known in the art as agglutination assays whereby a ligand is complexed with the reagent of this invention to form a detectable agglutination or -30~ 3 ~ ~3 ~
clumping of the pa~ticles. The resu:Lting agglutination can be detected in a variety of ways, for example visually or with suitable light scattering detection equipment. Representative agglutination techniques are described, for example, in US-A-4,419,453, US-A-4,808,524, US-A-4,828,978 and US-A-4,847,199.
Agglutination assays are prefe~ably carried out using reagents of the present invention which are detectably labeled in some manner, such as with a radioisotope in the particle or in the biologically active substance attached thereto, or with a colorimetric or fluorometric dye associated with the particle. Most preferably, a dye is within the 15 interior of the particle, that is away from its surface so as to not interfere with the attachment of a biologically active substance or its complexation.
Such particles can be core-shell particles having the dye within a core polymer while the shell copolymer is free of dye. This feature and methods of making such particles are described in more detail in US-A-4,808,524 and in EP-A-0 280 556. In core-shell polymer particles, the shell copolymer has a composition like that described herein (that is, having the necessary reactive carboxy groups), but the core polymer can be different and need not have reactive groups.
A method for the determination of an immunological species comprises:
A. contacting a specimen suspected of containing an immunological species with a reagent of this invention having a receptor for the species, to form a water-insoluble immunological complex of the species with the receptor, and B. after separating uncomplexed materials from the complex, detecting the presence of the complex as -31~ 3 ~ i3 an indicator of the presence or amount of the immunological species in the specimen.
The immunological species can be an antigenic material and the receptor an antibody therefor. Alternatively, the immunological species can be an antibody and the receptor an antigenic material specific therefor. Still again, the immunological species can be an antibody and the receptor an antibody specific therefor.
In still another embodiment, the reagent of this invention can be used in immunometric assays (often called "sandwich" assays). In such assays, the ligand of interest is complexed with two or more receptor molecules (the same or di~ferent), one of which is insolubilized or capable of being insolubilized (such as through an avidin-biotln bond~, and the other being water-soluble and appropriately labeled (such as with a radioisotope, enzyme, chemiluminescent moiety or other marker known in the art). For example, a sandwich assay for a ligand such as human chorionic gonadotropin (hCG) can be carried out with a reagent of this invention having antibodies to the hormone in combination with enzyme-labeled antibodies to hCG which will complex at different epitopic sites than the reagent antibodies. The resulting sandwich comple~ is insoluble, detectable and separatable frGm uncomplexed materials (such as with a microporous filtration membrane). In a preferred embodiment, the reagent of this invention has a receptor for the ligand of interest, and is immobilized on the membrane. Sandwich assays are well known in the art, including &B-A-2,074,727 and US-A-4,486,530, and references noted therein.
Preferably, in the sandwich assays, either prior to, simultaneously with or subsequently to the 32~
formation of the water-insoluble complex with the reagent of this invention, the ligand of interest is reacted with a water-soluble specific binding component specifically reactive therefor.
Other ligands which can be detected in sandwich assays according to this invention include, but are not limited to, Streptococcal antigens, antigens extracted from microorganisms associated with periodontal diseases, hepatitis antigens, HIV-I
and other retroviral antigens.
In one embodiment of the sandwich assay, the reagent of this invention is directly reacted with the ligand of interest, for example, where the ligand is an antigen, and the reagent comprises antibodies thereto. In another embodiment, however, the reagent is complexed with the ligand indirectly, that is, through an intermediate linking moiety. One example of this is shown in US-A-4,870,007 where complexation is through an avidin-biotin bond.
Another embodiment of this invention is what is known as a hybridization assay wherein a targeted nucleic acid is detected using complementary probes, one of which is suitably labeled, and the other is immobilized, or capable of being immobilized. The reagent of this invention can be used as an immobilized probe (also known as a capture probe~ in such assays. Examples of hybridization assays are shown, for example, in US A-4,358,535 and US-A-4,486,539. These reagents can also be used as capture probes after what is known in the art as polymerase chain reaction amp~ification, for example, as described in more detail in US-A-4,683,195, US-A-4,683,202 and Canadian Application Serial No.
2,002,076. An amplified nucleic acid is immobilized by hydridization with the reagent of thi~ invention.

~33~
In particular, a method for the detection of a nucleic acid comprises:
A. forming a water-insoluble hybridization product between a nucleic acid of interest, with a reagent of this invention having an oligonucleotide covalently attached to the particle through the reactive carboxy group or salt thereof, the oligonucleotide being substantially complementary to the nucleic acid of interest, and B. detecting the presence of the hybridization product as an indication of the presence or amount of the nucleic acid of interest.
In preferred hybridization assays, the nucleic acid of interest is amplified using polymerase chain reaction (known in the art) wlth suitable reagents (for example, DNA polymerase, dNTPs, primers) prior to capture with the reagent of this invention. ~IV-I DNA, cytomegaloviral DNA and ~-globin DNA are readily detected using amplification and detection according to this invention. In one embodiment, one of the primers is biotinylated, and detection of the ampliried nucleic acid is accomplished using a conjugate of avidin and an enzyme. The hybridized product can be captured using the reagent which may be attached to or localized on a substrate of some type, including a microporous substrate such as a membrane, or a compartment of a self-contained reaction pouch.
The analytical, sandwich and hybridization assays of this invention can be carried out using suitable equipment and procedures whereby complexed or hybridized product is captured or separated from uncomplexed materials by filtration, centrifugation or other means. Preferably, such assays are carried out using disposable test devices which contain microporous filtration membranes (for example those ,6,~

commercially available from Pall Corp.).
Representative test devices are shown in US-A-3,825,410, US-A-3,970,429 and US-A-4,446,232.
Particularly useful test devices are shown in Canadian Application Serial No. 563,473, and are commercially available as SurecellTM test devices ~Eastman Kodak Co.).
The analytical separation method of this invention can be used to isolate one or more analytes of interest from a mixture oi biological materials.
Thus, the Ieagent of this invention (or several reagents having different substances attached to particles) is generally placed in a column through which a fluid containing the mixture of biological materials is poured, allowing the reagent to extract from the fluid those materials one wants to isolate.
This may be useful in the purification of nucleic acids, enzymes, carbohydrates, proteins, lipids, vitamins, steroids, antibodies, peptides or hormones. This procedure is also known as affinity chromatogIaphy.
Affinity chromatography can also be used to concentrate dilute solutions of proteins in order to remove denatured ~orms thereof from refined proteins, and in the separation and resolution of protein and peptide components which have originated in specific chemical modifications.
Another use oX this method is to purify nucleic acids, such as those resulting from polymerase chain reaction amplification, as described, for example in Canadian Application Serial No. ~,011,818.
The reagent of this invention can be supplied for any of the described methods as a single material, or it can be supplied in an analytical element as described above, or yet again in ~ ~3 I1~ e~

combination with other reagents, test devices and equipment in a diagnostic test kit. For the purification method, the reagent can also be supplied in an affinity chromatography column.
Specifically, a kit for a hybridiæation assay includes a reagent of this invention having an oligonucleotide complementary to the nucleic acid of interest, and one or more other reagents (for example, labeled probe or po:lymerase chain reaction reagents), solutions (such als wash or extraction solutions) or articles (such as pipettes, filte~s, test devices or test vessels) needed for the assay.
In another embodiment, a kit useful or determination of a ligand (for example immunoassay, sandwich assay, diagnostic ~est or competitive binding assay) includes the reagent of this invention, and one or more other reagents, solutions or articles needed for such an assay (such as ligand analog, labeled receptor, dye-providing compositions, substrates, wash solutions, filters, test devices, extraction reagents and others known in the art).
In the analytical purification method of this invention, the reagent in the chromatography column captures one or more of the substances in the mixture of substances poured through the column.
In one embodiment, the predetermined substances are captured by the reagent, the original eluent is discarded and the captured substances are removed from the column using a solvent which alters the binding characteristics of the substances so they can be uncomplexed. Such solvents include buffers which alter the pH, salt solutions which alter the ionic nature of the complex or solutions containing a second species which will specifically bind ~o the reagent and replace the captured substance.

~3 ~`3~

Alternatively, the predetermined substances captured by the reagent are discarded, and other chemical or biological materials remaining in the original eluent are collected.
The following examples are for illustrative purposes only, and not to limit the scope of the invention. All percentages are by weight, unless otherwise specified.
Example 1: ReagQnts ~aving Labeled Bovine Gamma G~obulin This example illustrates the preparation of several reagents of this invention having a radio-labeled protein attached to the polymeric particles. Two different activating a~ents were used to prepare the reagents. The reagents were ccmpared to a Control reagent similarly prepared uging a copolymer outside the scope of this invention.
Copolymers prepared according to the procedures described in copending Canadian Patent Application (identified noted above) were used in this example. The copolymers were:
Test A: Poly(styrene-co-mono-2-methacryloy~o xyethyl glutarate) ~97.84:2.16 molar ratio), Test B: Poly(styrene-co-mono-_ ~
~-vinylbenxyl glutarate) (97.8:2.2 molar ratio), Test C: Poly[styrene-co-monomethacryloyl-penta(oxyethylene) glutarate]
(98.7:1.3 molar ratio), Control Poly(styrene-co-acrylic acid~
(95:5 molar ratio).
The protein was attached to the carboxy groups of these polymers using either of the carbamoylonium compounds: (a) 1 (4-morpholino-carbonyl)-4-(2-sulfoethyl)pyridinium hydroxide, inner 3 7 ~ ~ r 3 ~ ~ ~
salt, or (b) l-(l-pyrrolidinylcarbonyl)pyridinium chloride as activating agents. All polymers were treated under the same conditions. The final dispersions comprised 1% of 3H bovine gamma globulin per gram of polymeric particles, 0.3 mg of protein per 30 mg dry weight of particles i~
2-(4-morpholino)ethanesulfonlc acid buffer (0.1 molar, pH 5.5. The amount of activating agent was 16.6 mg of (a) or 9.6 mg of ~b) per tube.
These dispersions were prepared by putting the suspensions of particles (30 mg dry weight) in large microfuge tubes, and each was brought to a volume of 1.5 ml using the noted buffer. The resulting suspensions were centrifuged for 15 minutes at 14,000 rpm and the supernatant discarded. Buffer (1 ml, 0.1 molar) was added, followed by addition of a solution (300 ~1) of the act;vating agent to each tube. The solution of activating agent (a) was prepared by dissolving 199 mg in 3.6 ml of 0.1 molar buffer, and the solution of activating agent (b) was prepared by dissolving 115 mg in 3.6 ml of 0.1 molar buffer. The tubes were then capped and rotated end-over-end at room temperature for 10 minutes. A
solution (30 ~1) of the labeled protein (10 mg/ml) was added to each tube followed by rotation end-over-end for 4 hours at room temperature.
Reaction of the protein with the carboxy groups on the particles was quenched by the addition of bovine serum albumin (250 yl, 100 mg protein/ml) to each tube. The tubes were then rotated again for an additional 16 hours at room temperature, and each reaction mixture (~50 ~1) was removed to determine the total labeled protein.
A sample (500 ~1) of each reaction mixture was also removed and treated with buffer (400 ~1, 0.1 molar) and sodium dodecyl sulfate (100 yl of a 2 ~ .A.~ 3 10% solution in deionized distilled water). The resulting mixtures were mixed by tumbling at 37C for 16 hours on a rotating disc mounted at a 45 angle (the treatment with surfactant removed adsorbed, but not covalently bound, protein from the particles).
The reaction mixtures were centrifuged, and aliquots (500 ~1 each) were removed to determine the amount of free labeled protein.
The total of H bovine gamma globulin bound to the particles, the amount of H bovine gamma globulin covalently bound to the particles and the covalent/total bound rat:lo are shown in the followin~ Table I. The results show that the reagents prepared according to this invention acceptably bind antibody for use in immunoassays.

T A B L E
Test Activation Total % Covalent % Ratio Agent BoundBound A a 97 96 99 b 91 89 98 B a 97 97 100 b 94 93 99 C a 90 90 100 b 49 48 100 Control a 97 97 100 b 95 94 99 Example 2: Rea~ent Havin~ Anti-Thvroxine Antibodies This example illustrates the preparation of a reagent of this invention having antibody molecules to thyroxine covalently attached to polymeric particles.
The procedure described in Example 1 was used to attach monoclonal anti-thyroxine antibodies (available from Cambridge/Ventrex Laboratories, Inc.) 2~3~

to the same copolymers, except that the antibody solution (130 ~ .3 mg protein/ml) was used in place of labeled bovine gamma globulin. Both activating agents (a) and (b) were used to prepare the reagents. The reaction was quenched, the reaction mixtures were centrifuged, the supernatants decanted, and the par~icles resuspended in phosphate buffered saline solution (1 ml, p~ 7.4). This step was repeated four times, and during the last time, the solids were resuspended in phosphate buffered saline solution (1.8 ml) ahd merthiolate preservative (0.02%) was added.
The relative amounts of active antibody in the resulting dispersions were determined in an assay in which serial dilutions of the reagent dispersions were mixed with a fixed concentration of an enzyme-labeled analog of thyroxine and alkaline phosphatase (prepared as described by Ito et al in Clin.Chem., 30(10), pp. 1682-1685, 1984). The dilutions were incubated for about one hour with constant agitation at room temperature in phosphate buffered saline solution containing bovine serum albumin (1%). The amount of labeled analog remaining in solution after centrifugation was determined, and the concentration of thyroxine binding sites required to blnd 50% of the analog was calculated. The results are summar;zed as follows in Table II. These data show that the reagents of this invention bind the labeled analog at considerably lower (3-5 times) concentration than the Control reagent using either activating agent.

2 ~ 9 T A B L ~ II
Theoretical Thyroxine Binding Sites Where 50%
of ~bel is Bound (n~Q~E~
~g_ t (a~ A~ent (b) Test A 20 10 Test B 23 13 Test C 36 16 Control 100 56 Example 3: ~Q3gent Eaving Oligonucleotide Convalently ~ound to Particle~
This example illustrates the preparation of a reagent of this invention having an oligonucleotide (or nucleic acid~ which is complementary to a nucleic acid of interest.
A sample (50 mg, 0.379 ml of 13.18%
suspension) of poly(styrene-co-mono-m & ~-vinylbenzyl glutarate) (97.8:2.2 molar ratio) particles was added 2~ to 2-(4-morpholino)ethanesulfonic acid buffer (5 ml, 0.1 molar, pH 6). The resulting suspension was centrifuged at 3200 rpm to pelletize the polymer particles. After the supernatant was decanted, the particles were resuspended in buffer (5 ml) in a centrifuge tube. Added to this was the activating agent, N-[3-(dimethylamino)propyl]-N'-ethyl-carbodiimide hydrochloride (18.8 mg), followed by an oligonucleotide (0.625 optical densi~y, 1.875 nmole) having the following sequence complementary to 30 n - globin DNA:

5'-X-CCTCAAACAGACACCATGGTGCACCTGACTC~3' wherein A, C, G, and T are the standard representations for adenine, cytosine, guanine and tyrosine bases in the nucleic acid sequence, and X

represents a tetraethylene glycol amine linker having the structure:
o H2N t CH2CH2034 1- O-ONa and attached according to the teaching of US-A-4,914,210 (noted abo~e). The tuhe was capped and rotated end-over-end $or 18 hours at room ; 10 temperature.
The suspension was then centrifuged at 3200 rpm, the supernatan~ decanted, and the solids resuspended in glycine (0.1 molar, pH 8.5~ containing merthiolate (0.01%). This wash procedure was repeated twice. The final suspension contained 0.~7%
solids of reagent as determined using spectrophotometric light scattering.
Example 4: Reagent Having Antibodies tQ
Euman Chorionic Gona otropin This example is similar to Example 2. I~
illustrates the preparation of a reagent useful in an immunoassay.
A sample (10 mg, 65.8 ~1 of a 15.2%
suspension) of polymeric particles as described in Example 3 was added to 2-(4-morpholino)ethanesul~onic acid buffer (1 ml, 0.1 molar, p~ 6~. The resulting suspension was centrifuged at 3200 rpm to pelletixe the poLymer particles. A~ter the supernatant was decanted, the particles were resuspended in buffer (1 ml) in a centrifuge tube. Added to this was the activating agent, N-t3 (dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride (3.76 mg). The tube was capped and rotated end-over-end for 10 minutes at room temperature. Affinity purified goat antibodies to the beta subunit of human chorionic gonadotropin (hCG~ (O.:L23 mg, OEM Concepts, Toms River, N.J.) were added to the tube, and it was rotated again for 18 hours. The suspension was centrifuged at 3200 rpm, the supernatant discarded, and the pellet resuspended in glycine (0.1 molar, pH 8.5) containing merthiolate ~0.01%). This procedure was repeated twice to provide a reagent of this invention having antibodies covalently attached to the particles.
~xamples 5-7: Reagents Havin~ Antibodies to P enobarbital. Phenytoin and Digoxin These examples illustrate the preparation of reagents of this invention which are useful in both solution and dry competitive binding assays.
Suspensions of polymeric particles used in Example 1 were used [Tests A-C and the Control (except the Control polymer particles had a 96.65:3.35 molar ratio)~. The activating agent used was l-(l-pyrrolidinylcarbonyl)pyridinium chloride.
All particles were treated similarly. The final dispersions comprised antibody (anti-phenytoin for Example 5, anti-phenobarbital for Example 6 and anti-digoxin for Example 7), 0.3 mg of protein per 30 mg dry weight of particles in 2-(4-morpholino)-ethanesulfonic acid buffPr (0.1 molar, pH 5.5, and activating agent (1.5 mmole agent/g particles, that is 0.045 mmole).
These dispersions were prepared by putting suspensions of particles (30 mg dry weight) in large microfuge tubes, and each was brought to a volume o~
1.5 ml using the noted buffer. The resulting suspensions were centrifuged for 15 minutes at 13,000 rpm and the supernatants discarded. Buffer (~ ml, 0.1 molar) was added to each tube, followed by addition of the activating agent (0.3 ml of a solution at 0.15 molar in buffer). The tubes were then capped and rotated end~-over~end at room temperature for 10 minutes. A solution of the antibodies was added to the respective tubes-: 0.07 ml of anti-phenytoin (4.3 mg/ml), 0.115 ml of anti-phenobarbital (2.6 mg/ml) and 0.196 ml of anti-digoxin (1.53 mgtml), 0.3 mg total antibody for each, followed by rotation end-o~er-end for 4 hours at room temperature.
Reactions of the antibodies with the activated particles was quenched by the addition of bovine serum albumin (300 ~1, lO0 mg protein/ml) to each tube. The tubes were then rotated again for an additional 16 hours at room temperature, centri~uged and supernatants removed for later ELISA analysis, the particles resuspended in phosphate buffered saline solution (1 ml, pH 7.4). This step was repeated three times, and the final resuspension was in phosphate buffered saline solution (1.8 ml) containing merthiolate (0.02%).
The supernatants from the reaction mixtures were analyzed for total antibody concentration by ELISA. The amount of antibody covalently bound to the particles was calculated from the ELISA results.
The relative amounts of active antibody in the preparations were determined in an assay in which serial dilutions of the reagents were mixed with fixed concentrations of the conjugates: alkaline phosphatase-labeled phenytoin, alkaline phosphatase-labeled phenobarbital or horseradish peroxidase-labeled digoxin, all of which were prepared using known procedures as described by Erlanger et al, J.Biol.Chem., 234, 1090 (1959). The reaction between the diluted reagent dispersions and the enzyme-labeled analogs were incubated for one hour at room temperature with constant agitation in phosphate buffered saline solution containing 0.1 or 1% bovine serum albumin. The amount of enzyme-labeled analog remaining in the supernatants _44_ ~ L~ ~3 ,~
after centrifugation was determined, and the concentration of antibody-binding sites required to bind 50% of the enzyme labeled analog was calculated. The results are summarized as ~ollows in Table III:

T A B L E III
Theoretical Binding Sites for Binding 50% of Labeled Analog (nmolar) 10 Test Phenytoin Phenobarbital Digoxin Example 5 4.5 6.0 0.83 Example 6 5.1 7.1 0.53 Example 7 7.0 6.0 6.3 Control 6.3 10.0 5.
lS The results indicate that each of the reagents of Examples 5 and 6 has higher activity than the same antibody immobilized as part of the Control because they required less reagent for binding 50% of the binding sites. In Example 7~ the phenobarbital reagent exhibited better activity than the Control, but the other two reagents had slightly less activity than the Control.
Examples 8-9: Reagents ~avin~ Iodinated Proteins These examples show the preparation of reagents of this invention having iodinated bovine gamma globulin covalently attached to polymeric particles.
All particles were treated similarly, the final reaction dispersions contained 125I-bovine gamma globulin (0.3 mg?, polymeric particles (30 mg dry weight), l-(l-pyrrolidinylcarbonyl>pyridinium chloride activating agent (0.5 mmole/g particles) in 2-(4-morpholino)ethanesulfonic acid buffer (0.1 molar, pH 5.5).
'rhe polymeric particles used were those identified as Test B (Example 8), Test C (Example 9) -45~
and the Control o$ Example 1 above. Aliquots of the particle dispersions were placed in 2 ml microfuge tubes and buffer (0.1 molar) added to a total volume of 1.5 ml. The dispersions were centrifuged 10 minutes at 13, ono rpm and the supernatants discarded. The beads were redispersed in the buffer (1 ml) and the ~ctivating agent (0.3 ml, 0.15 molar). The tubes were capped and rotated end-over-end for 10 minutes. A solution of the iodinated protein was added to provide 0.3 mg tota~
in each tube, and one set of the reaction mixtures were rotated end-over-end at room temperature for 6 hours, while the other set was rotated end-over-end at room temperature for 52 hours. The reaction mixtures were centrifuged and the supernatants removed and analyzed for radioactivity. The resulting reagents were resuspended in phosphate buffered saline solution (1 ml), centrifuged, resuspended in sodium dodecyl sulfate ~1%), and analyzed for radioactivity to determine the total amount of iodinated protein bound to the particles.
The particles were then incubated for 16 hours in sodium dodecyl sulfate (1%) at room temperature while tumbling end-over-end to removed adsorbed protein~ but leaving covalently bound protein attached. The reagents were pelletized, the supernatants removed and analyzed for radioactivity, then resuspended in sodium dodecyl sulfate (1%). The reagents were centrifuged again, the supernatants discarded, and the reagents analyzed for radioactivity.
The percent of iodinated protein covalently bound to the particles for each reagent are noted as follows in Table IV.

2~4~

T A B L E IV
Test ~/O Cova]entlv Bound _h~E~ 52 hpurs Example 8 96 100 Example 9 45 100 Control B0 100 These results indicate that the polymeric particles used in Example 8 react more quickly with the protein than do the Control particles so that a shorter time is needed for complete seaetion of protein with the particles. This represents a manufacturing advantage. It also represents an advantage in that antibodies which may be sensitive to the conditions needed for attachment will less likely be deactivated by those conditions due to the shorter reaction times. After 6 hours of reaction, the particles of Example 9 do not provide results as good as the Control, but this example demonstrates that it is still a useful reagent. Longer times ~or reaction of protein with the particle carboxy groups results in complete attachment for all the particles. This indicates that different polymers and proteins may require various reaction conditions ~5 to prepare desired reagents, aæ one skilled in the art would readily understand.
Example 10: preparation of Reagents Having QligQ-nucleotides for Cytomegalovirus DNA
This example demonstrates the preparation of reagents having an oligonucleotide covalently attached to carboxy-containing copolymers as described herein. The oligonucleotide is directed to a nucleic acid sequence of cytomegalovirus DN~.
The oligonucleotide used herein was 35~ synthesized using a Biosearch 8650 DNA synthesizer by the phosphoramidite method described in 3 ~ ~

US-A-4,725,677 with some modifications in activator ~(Activator Gold available from Beckman), an additional aqueous wash step (before oxidation) and l-methylimidazole as a capping reagent in place of 4-(N,N-dimethyl- amino)pyridine. The reagents used for derivatizing the oligonucleotide for addiDg linkers and spacers are described in US-A-4,914,210.
The oligonucleotide reagents were designed with either (1) a single amino linker at the 3'-end (identifi.ed as LB09), or (2) with an amino linker and two tetraethylene glycol spacers at the 3'-end (identified as LB08). The oligonucleotide sequence is as follows using standard base identification (A, C, G, and T):
5~-TCACCCCCA~ AGTCCCCTGT ACCC-X-3' wherein, for LB08, X represents an amine linker connected to two tetraethylene glycol spacer units, and, for LB09, X represents the same amine linker without spacer units having the structure:

R

H2NtCH2CHCH2--O--P--O--O ONa O=P-O-Thymidine ONa The 5'-end of the purified, deblocked oligonucleotides were labeled with 32p by Xinasing with ~32P-ATP. LB08 and LB09 ~2.0 OD (260 nm) units of each in 20 ~1] were labeled and used directly in the tests. The LB08 and LB09 oligonucleotides (2 ~1) were added to activated bead samples as indicated herein.

` 2Q-~3~

The polymeric particles used in this example were as follows:
Polymer A Poly(styrene-co-mono-2-methacryloylo xyethyl glutarate) (97.8:2.2 molar ratio).
Polymer B Poly[styrene-co-mono-m &
p-(60:40)--vinylbenzyl glutarate]
97.7:2.2 molar ratio).
Polymer C Poly~styrene-co-monomethacryloyl-penta(oxyethylene)glutarate]
(98.7:1.3 molar ratio).
Polymer D Poly[styrene-c -monomethacryloyL-deca(oxyethylene) glutarate]
(98.3:1.7 molar ratio~.
Polymer E Poly[styrene-co-mono-2-(m &
~(60:40)vinylbenzylthio)ethyl glutarate] (98.3:1.7 molar ratio).
Polymer F Poly(styrene-co-mono-~-vinylbenzyl glutarate) (97.Z:2.2 molar ratio).
~0 Polymer G poly[styrene-co-mono-2-(~-~inyl-benzylthio)ethyl glutarate]
(98.3:1.7 molar ratio).
Polymer H Poly[styrene-co-3 (~-vinylbenzyl-thio)propionic acid] (97.6:2.4 molar ratio).
Control A Poly(styrene-co-acrylic acid) (97.5:2.5 molar ratio).
Control B Poly(styrene-co-3-acrylamido-3-methylbutanoic acid) (96.9:3.1 3~ molar ratio).
The particles were provided as aqueous la~ex dispersions having the % solids indicated in the Table V below. Attachment of the oligonucleotides was accomp:Lished by using either of two acti~ating agents: (a) N-(3-(N,N-dimethylamino)propyl)-N'-ethylcarbodiimide hydrochloride (either 100 ~1 of a 2 ~ 9 170 mg/1.35 ml solution for Tests 1-6 or 100 ~1 of a 538 mg/ml solution for Tests 7-16), and ~b) l-(l-pyrrolidinylcarbonyl)pyridinium chloride (100 ~1 of 538 mg/ml solution). The activating agent was added to a sample of each dispersion and allowed to react for either 10 minutes (Tests 1-6) OI 25 minutes (Tests 7-16).
The resulting activated polymeric particles were then treated with either labeled oligonucleotide (LB08 or LB09, 2.0 ~1), and Tests 1-6 were allowed to react for one hour after mixing while Tests 7-16 were allowed to react for 2.5 hours ater mi~ing.
The bound oligonucleotides were then separated by centrifugation (2 minutes), and the supernatants were decanted for radioactive counting. The resulting reagents were washed with water, resuspended and centrifuged three times. The combined supernatants from each test and the resuspended reagents were then evaluated for radioactivity, and the results are presented in Table V below.
The results indicate that the amount (~/O~ of oligonucleotide covalently bound to the particles is improved for the reagents of this invention as compared to the Control reagents. Some tests showed much more improvement than others, while some reagents showed improvement with certain activating agents and not others. These data would suggest to one skilled in the art how to find a substantially improved reagent for the detection of cytomegalovirus DNA.

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Example 11: AssaY for Human Chor~ Gonadot~o~in ThiS example demonstrates the practice of the present invention to detect hCG uæing a reagent of this invention.
Materi~ls:
SurecellTM disposable test devices were used containing LoProdyneTM microporous membranes (5 ~m, Pall Corp.), each coat:ed with FluoradT~ FC
135 nonioDic surfactant (3M, 0.05 g/m2~.
A reagent of this invention was prepared by covalently attaching affinity purified goat anti-hCG
alpha polyclonal antibodies (OEM Concepts, Toms River, N.J.) to particles o~ poly[styrene~ 3-(~-vinylbenzylthio)propionic acid] (97.6:2.4 molar ratio) using l~ pyrrolidinylcarbonyl)pyridinium chloride as the activating agent. The procedure for attaching the antibodies was as ~ollows:
A suspension of polymeric particles (3%
solids) was mixed with the activating agent (0.1 molar) in 2-(4-morpholino)ethanesulfonic acid buffer in a microfuge tube. The tube was capped, and rotated end-over-end at room temperature for 10 minutes. A solution (810 yl) of the polyclonal antibodies (13.2 mg/ml) was added to the tube followed by rotation end-over-end for 18 hours at room temperature. The resulting reagent had about 99% of theoretical antibody covalently bound to the polymeric particles.
A composition (2 ~1) comprising the reagent (0.9%), polyacrylamide (5%), ~vitexTM dye (0.01%) and thimerosal preservative (O. 01%) in glycine buffer (0.1 molar, pH 8.5) was deposited on a finite area of the membrane in one of the test wells (designated ~he sample well).
Goat gamma globulin was eovalently bound to the same type of particles using the same procedure ~4~ 3 ~ ~ ~
and the resulting reagent was deposited onto the membrane in another test well (designated the negative control well). A third test well (designated the positive control well) contained anti-hCG antibodies covalently bound to the same type of particles and hCG antigen prebound to the antibodies.
The test solution contained hCG (50 mI.U./ml) in a solution of phosphate buffered saline solution (150 mmolar sodium chloride, 50 m~olar sodium phosphate, pH ~.2), hovine serum albumin (0.7%) and merthiolate (0.01%).
A conjugate of anti-hCG monoclonal antibodies (Cambridge Medical Diagnostics) and horseradish peroxidase (Miles) was prepared using ~he procedures described by Yoshitake et al, Eur.J.Biochem., 101, 395 (1979). This conjugate was ~mixed with Medi~ Peroxidase Diluent (Medix Biotech, Inc., Foster City, California) containing LonzaineTM C amphoteric surfactant (0.1%, Lonza Corp.). The final conjugate concentration was 3.38 ~g/ml.
A wash solution was prepared from sodium phosphate (0.1 molar, pH 7.2), sodium decyl sulfate (100 mmolar, 2.7%), sodium chloride (0.3 molar) and thimerosal (0.01%).
A dye-providing composition comprised 2-(4-hydroxy-3-methoxyphenyl)-4,5-bis(4-methoxyphenyl)-imidazole leuco dye (0.005%), poly(vinyl pyrrolidone) (1%), sodium phosphate buffer (5 mmolar, pH 6.8) diethylenetriaminepentaacetic acid (10 ~molar), 4'-hydroxyacetanilide (2 mmolar) and hydrogen peroxide (10 mmolar).
Assav Procedure:
The test solution (150 ~l) containing hCG
(50 mI.U./ml) was added to the three test wells of . 2~-~3~3~9 the test device, and the fluids were allowed to drain through the membranes. The buffered composition containing labeled antibody (1 drop, about 40 ~1) was added to each test well, and allowed to drain through. The test wells werle washed twice (each time with 300 lll) and allowed to drain through. The dye-providing composition (50 ~1) was then added to each test well and allowed to drain through. After incubation for less than 1 minute at room temperature, the dye density on the membranes was evaluated against a color chart for dye density with 10 representing the highest density. The area~
around the applied compositions in the test wells were evaluated as background. The assay was carried out three times.
The results are provided in the following Table VI as visual dye densities seen in the specific test wells for each of the three-tests:
T A B L E VI 0 Nega~ive Control Positive Control Well Sample Well Well Test Back round Test Background Test Background 2~ o 0 ~ 0 7 0 These data show that in three separate assays, a very low concentration of hCG (~0 mI.U.~
can be readily detected with zero background using the reagent of this invention;
For comparison, the above example was r~peated but using a Control reagent prepared with the polyclonal antibodies covalently attached to particles similarly prepared from poly[styrene-co-~-& ~-~2-chloroethylsulfonylmethyl)styrene] (95.5:4.5 molar ratio~ (see the teaching in EP-A-0 323 692, -56~ 3 ~ ~3 ~i~
published July 12, 1989). The antibodie6 were attached by mixing a suspension of the Control particles (3% solids) with borate buffer (0.1 molar, pH 8.5) in a microfuge tube. A solution (810 ~1) o~ the polyclonal antibodies (13.2 mg/ml) was added to the tube followed by rotat:ion end-over-end for 18 hours at room temperature. The resulting reagent had about 99% of theoretical antibody covalently bound to the particles.
The test solutions containing hCG, conjugate composition, wash solution and dye-providing composition were the same as described above.
The results of the Control assay using the procedure noted above were found to be as follows:
There was less dye formation generated by the low concentration (50 mI.U./ml) of hCG, and negligible background. Because the assay was carried out in less than two minutes, the dye signal for eight separate tests averaged only 1.9, which is considerably lower than the results provided with the present invention ~Table VI).
~xample 12: Reagent and Assav for HIV-I
DNA Detection This example illustrates the preparation of several reagents of this invention having oligonucleotides covalently bound to polymeric particles, and their use in nucleic acid assays to detect either HIV-I DNA, ~-globin DNA or both. The methods are carried out using analytical procedures and devices descri~ed in more detail in Canadian Application Serial No. 2,026,573 whereby the reagents were immobilized on LoProdyneTM microporous membranes (Pall Corp., 5 ~m).
Materi~ls:
The polymers used to prepare reagen~s for evaluation are as follows:

-57~ 3 ~ ~ ~
Control Poly(styrene-co-acrylic acid) (97.5:2.5 molar ratio).
Polymer A Poly(styrene-co-mono-m ~
~(60:40)-vinylbenzyl glutarate) (97.B4:2.:L6 molar ratio).
Polymer B Poly[styrene-co-3-(~-vinylbenzyl-thio)propionic acid] (97.59:2.41 molar ratio).
Polymer C Poly[styrene-co mono 2-(4-vinyl-benzylthio)ethyl succinate]
(98.17:1.,33 molar ratio).
Polymer D Poly(styrlene-ço-mono-4-vinylbenzyl succinate) (97.71:2.29 molar ratio).
These polymers were prepared using the procedures described in more detail in copending Canadian Patent Application (identified above).
The oligonucleotide used to make the reagent is complementary to a portion of EIV-I DNA in the g~
region, and has the following nucleic acid sequence:
S'-X-ATTAAATAAA ATAGTAAGAA T-3' wherein X represents an amino group attached to the oligonucleotide through an ethylene glycol spacer according to US-A-4.914,~10.
Suspensions of particles of each polymer were washed with 2-(N-morpholino)ethanesulfonic acid buffer (0.1 molar, pH 6). Samples (30 ~g) of particles were suspended in the buffer (1 ml) and mixed with the oligonucleotide (0.0288 ml of 57.3 OD/ml purified water, 1.65 OD units) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.15 ml of 100 mg/ml buffer solution). The resulting mixtures were rotated end-over-end at room temperature for 15 hour~ and centrifuged. The reagents were washed three times -58~ f~
with purified water and resuspended in purified water at a 0.9% solids content.
The resulting reagents were deposited on separate LoProdyneTM microporous membranes located 5 in test wells o~ SurecellTM disposable tes-t devices, in de~ined regions less than 2 mm2 in diameter, and allowed to dry to about 30 minutes at room temperature. The resulting diagnostic test elements were then used in t:he assays described below.
Primers used in the amplification of XIV-I
DNA had the following nucleic acid se~uences:
5'-X-TTTGGTCCTT GTCTTATGTC CAGAATGC-3' and 5~-ATAATCCACC TATCCCAGTA GGAGAAAT-3' wherein X represents a biotintetraethylene glycol ester spacer, prepared and attached according to US-A-4,914,210.
Primers used in the amplification of ~-globin DNA had the following nucleic acid se~uences:
5'-X-CAACTTCATC CACGTTCACC-3' and 5l ACACAACTGT GTTCACTAGC-3' wherein X represents a biotin amino linker, prepared and attached according to US-4,914,210.
All primers and oligonucleotides used in this example (and in all other examples of this application) were prepared using standard phosphoramidite chemistry, puri~ied by high pressure liquid chromatography and characterized by standard sequencing procedures.
DNA polymerase was isolated from Thermus a~uaticus according to procedures described in US-A-4,889,818 (1 unit corresponds to 10 mmole of dNTP incorporated into the primer extension product in 30 minutes at 37~C~.
A streptavidin-horseradish peroxidase conjugate was obtained from Zymed Labs (San Francisco), and was diluted 1:4000 with a phosphate -5~- 2~
buffered saline solution containing casein (0.5~
3-(N-morpholino~propanesulfonic acid buffer (100 mmolar, pH 7.5) and pxeservative (O.OlV/o~. The final conjugate concentration was 312 ng/ml. The phosphate buffered saline solution contained sodium phosphate (25 mmolar, pH 7.3) and sodium chloride (75 mmolar).
A dye-providing composition comprised 2-(4-hydroxy-3-methoxyphenyl)-4,5~-bis(4-methoxyphenyl)-imidazole leuco dye (0.005%), poly(vinyl pyrrolidone) (1/~), sodium phosphate buffer (5 mmolar, pH 6.8) diethylenetriaminepentaacetic acid (10 ~molar), 4'-hydroxyacetanilide (5 mmolar) and hydrogen peroxide (10 mmolar).
AssaY:
The procedure for detecting HIV-I was carried out in the following manner.
An ~IV-I DNA target was isolated from the HUT cell line, which contains a single integrated copy of the HIV viral genome, obtained from Dr.
Bernie Poiesz at Syracuse University.
A ~-globin DNA target was isolated from human placental cells obtained from Sigma Chemical Co.
Three target samples were subjected to amplification and detection procedures:
Sample A~ Contained ~IV-I DNA target, ~-globin DNA target and the primers for the HIV-I DNA
target only.
Sample B: Contained ~-globin DNA target and primers therefor only.
Sample C: Contained both targets and primers for both targets.
Mixtures for polymerase chain reaction to amplify the target HIV-I DNA contained tris(hydroxy-methyl)aminomethane buffer ~10 mmolar, pH 8), potassium chloride (50 mmolar), magnesium chloride (10 mmolar), gelatin (10 ~g), the appropriate ? l~

primers noted above ~100 pmolar of each), dNTP'~ (1.5 mmolar of each), the DNA polymerase noted above (7.5 units), and target: either ~-globin DNA (1 ~g) or ~IV-I DNA (about 10 16 molar). The total volume of each mixture was 100 ~1.
Each reaction mixture was placed into a polypropylene microcentrifuge tube, primer extension products were formed and amplification of the target nucleic acid was carried out using 30 thermal cycles as follows:
70C, rising to 95C l minute 95C 0.5 minute 95C, lowering to 55C 1.25 minute 55C 0.5 minute 55C, rising to 70C 0.75 minute, and 70C 1 minute.
After amplification, aliquots (5 ~l) of each reaction mixture was added to a solution (95 ~1~ containing tris(hydroxymethyl)aminomethane buffer (lO mmolar, pH 8~, potassium chloride (50 mmolar), magnesium chloride (lO mmolar) and gelatin (l ~g/10 ml solution), heat denatured (5 minutes at 95C) and added (about 95 ~1 of each mixture in each test well) to the SurecellTM test devices described above having reagent immobilized therein.
Each test well was sealed with tape, and the devices were incubated at 42C for 5 minutes to hybridize the amplified target EIV-I DNA to the water-insoluble reagent immobilized in the test wells. The sealing tape was removed from each test well, followed by washing with a buffered solution (250 ~1) containing phosphate buffer (20 mmolar, pH
7.4), sodium chloride (300 mmolar) and ethylenediaminetetraacetic acid (2 mmolar) at 55C.
The peroxidase conjugate (50 ~1, 15.6 ng) was added to each test well, and the test devices -61- 2 ~
were incubated at room temperature for 2 minutes. A
second wash (250 ~l) was carried out using the buffered solution noted above. The dye-providing composition (lOO ~l) was then added to each test well, followed by another incubation at room temperature for 2 minutes. Dye formation was stopped by the addition of sodium azide (lOO ~l of 0.1%
solution), and the resulting red dye in the test wells was visually evaluated. Each assay was duplicated.
The results of dye :eormation was ~raded on a scale of O to lO with lO representing the highest dye density. The results in the following Table VII are duplicate readings for each reagent and sample.
T A B L E VII
Dve Dens itY
Sample A Sample ~Sample C
(HIV-I (B-Globin (~IV-I ~
~ Reagent DNA Only) DNA Only~~-Globin DNA) Control 2.0, 2.0 0.25, 0.254.0, 2.0 Polymer A 4.0, 3.0 0.25, 0.255.0, 4.0 Polymer B 6.0, 5.0 0.50, O.SO6.0, 6.5 Control 2.0, 4.0 O, 03.0, 3.0 Polymer A 3.0, 5.0 0.50, 0.254.0, 4.0 Polymer C 7.0, 7.0 0.50, 0.256.0, 7.5 Control 3.0, 2.0 0.5, 0.54.0, 2.0 Polymer A 4.0, 4.0 0.5, 0.55.0, 4.0 Polymer D 7.0, 7.0 0 5, 0.57.0, 6.0 These results indicate that the reagent of this invention was successfully used as a probe for the detection of amplified HIV-I DNA. The B-globin DNA target, however, was not detected to an ~ 3 appreciable extend even after its ampl;fication because no capture reagent was used having an oligonucleotide complementary to ~-globin DNA. The background dye densities (Sample ~) were acceptably low.
Example 13: B~ent and Ass~s for HIV - I DNA
and Beta-Globi:n DNA Detection This example was carried out similarly to Example 12 except a different oligonucleotide was used in prepaIing the reagent for HIV-I DNA
detection, and ~-globin DNA was also detected after amplification of the target nucleic acid using a reagent having an oligonucleotide complementary to ~-globin DNA.
The reagent for detecting ~IV-I DNA was prepared using an oligonucleotide having the following sequence:

5 ~--X--ATCCTGGGAT TAAATAAAAT AGTMGMTG TATAGCCCTA C--3 ' ~0 wherein X is an amino group attached to the oligonucleotide with a spacer using the procedure described in US-A-4,914,210.
The reagent for ~-globin DNA detection was prepared using an oligonucleotide having the following sequence:

5 '--X--CCTCAAACAG ACACCATGGT GCACCTGACT C--3 wherein X is an amino group attached to the oligonucleotide using the procedure described in US - A - 4, 914,210 (noted above).
Three target samples were subjected to amplification and detection procedures using the 35 HIV-I DNA reagent and the ~-globin DNA reagent:

-63- 2 ~ I 3 ~ ~ ~
Sample A: Contained HIV-I DNA target, ~-globin DNA target and the primers ~or the HIV-I DNA
target only.
Sample B: Contained ~-globin DNA target and primers therefor only.
Sample C: Contained both targets and primers for both targets.
Amplification was carried out as described in Example 12, and the resulting dye densities were evaluated as shown therein. The test results are provided in Table VIII below for each reagent and test sample.

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~xample 14: As say for HIV-I DNA in PQlvm-eri-c _ouch This example is similar to Example 12 except that HIV-I DNA is detected using a formulated containeI useful for holding reagents for the assay.
The container is similar in const~uction to that described in copending Canad:ian Application Serial No. 610,728.
Materials:
A container for the assay was prepared from two sheets of Scotchpak 241T~ heat sealable ilm (3M Corp.~ which is a laminate of polyethylene on poly(ethylene terephthalate). The polyethylene side of the film served as a heat-activated adhesive.
flow channel was formed in one of the ~heets by laying it on a mold having a groove therein, heating it at a temperature below the melting point of the polyethylene, and while it was heat relaxed, mechanically stretching the film into the groove to form a channel. The channelled piece was then aligned with a second piece of Scotchpak 241TM heat sealable film on which a reagent of this invention (described below) was immobilized in a particular region. This al;gnment was with the polyethylene surfaces of the two films facing each other. The two films were then heat-sealed along both side boundaries of the channel so there was no leakage along those sides.
The resulting l'detection channel'l was used to simulate a "flow-by" assay format. Each detec~ion channel had an inlet port for sample introduction, a reagent of this invention immobilized therein, and an outlet port to allow fluids to exit the channel and to be collected. Fluid reagents were forced into the channel to contact the immobilized reagent before exiting the channel.

The reagents used in this example were ~ ~ 3 prepared using Polymers B, C and D of Example 12, and were prepared using the procedure of that example.
The resulting reagents (2 ~1 of a 0.9% suspension) were deposited on a defined region (about 2 mm diameter) of the detection channel for individual pouches as described above (channel size was about 19 mm by 8 mm) and allowed to dry at room temperature.
The HIV-I DNA target and primers were the same as those used in Example 12. The reaction mixture for polymerase chain reaction was the same except gelatin was present at 0. Ol~/o and the primers were present at 1 ~molar each.
Assay:
A solution containing the ~IV-I DNA target (10 16 molar) was amplified using polymerase chain blisters on a standard thermocycler and the polymerase chain reaction mixture (100 ~1) for 30-33 cycles using the protocol:
incubation at 94C 4 seconds, and incubation at 65C 30 seconds.
A sample (200 ~1~ of a 5:95 dilution of the resulting amplified reaction mixture in a buffer solution [containing tris(hydroxymethyl~aminomethane buffer (10 mmolar, pH 8.3), potassium chloride (50 mmolar~, magnesium chloride (10 mmolar) and gelatin (0.01%)] was heated in a tube at 95C for 5 minutes to denature the double-stranded targets. The heated solution was transferred to a syringe barrel and injected into each detection channel in a manner to insure even coverage of the immobilized reagents in the channels. Each channel was then incubated at 42OC for 5 minutes to anneal the reagent to the single-stranded HIV-I nucleic acid target. The fluid was removed from the detection channels by forcing the liquid out with air and the fluid was collected with a syringe.

~ 13~o~

A preheated (55C) wash solution was injected into each detection channel. This solution comprised a buffer solution (400 ~l) comprising sodium dihydrogen phosphate (10 mmolar, pH 7.4), sodium chloride (150 mmolar), ethylenediamine~etraacetic acid (1 mmolar) and sodium decyl sulfate (1%). The wash solution was removed, and the streptavidin-horseradish peroxidase conjugate of Example 12 (200 ~1) was then injected into each detection channel and incubated at room temperature for two minutes. The fluid was re~oved and a æecond wash (400 ~1) was introduced and removed. The dye-providing composition of Example 12 (200 ~l) was then introduced into each channel and incubated at room temperature for 1-2 minutes and removed.
Lastly, a solution of sodium azide (200 ~1 of a 0.1% solution) was introduced to stop dye formation, and removed.
The resulting dye formed in each channel was visually graded on a scale of 0 to 10 with 10 representing the highest dye density. The results o~
the readings are shown in the following Table IX for the average of three separate tests for each reagent.

T A B L E IX
Reagent Dye Density Polymer B 5.2 Polymer C 5.5 Polymer D 5.3 Similar experiments were carried out successfully wherein the container in which the dye was formed was a self-contained vessel (or pouch) which had compartments for all rea~ents and solutions needed for the assay, and a separate compartment for the reaction. Such self-contained vessels are -68- 2 ~ t~
described in more detail in Canadian Application Serial No. 610,728.
Example 15: Prepara n of Re~gents Us ful ~or Detecting Cvtomegaloviral DNA
This invention describes a novel method for preparing hybridization reagents which can be used in hybridization or polymerase chain reaction assays.
The reagents are useful in the detection of cytomegaloviral DNA.
Materials:
Polymeric particles composed of poly-[styrene-co-3~ vinylbenzylthio)propionic acid]
~97.6:2.4 molar ratio, 1.4 ~Im average diameter) were used to make the reagents.
The activating agent was 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride, and the buffer was 2-(N-morpholino)ethanesulfonic acid (0.1 molar, pH 6).
The oligonucleotides used have the following sequences, identified using standard notation for the bases:
#1 5'-GGTGTCACCC CCAGAGTCCC CTGTACCCGG-3' ~2 5'-GACACAGTGT CCTCCCGCTC CTCCTGAGCA-3' #3 5'-GTGG MGGCG GCTCGCTGGA AGCCGGTCGT-3' #4 5'-GAACCGAGGG CCGGCTCACC TCTATGTTGG-3~
The oligonucleotides were attached to the particles through the 3'-end using the following linking group:
O O
Il 11 H2N-CH2fHCH2-0-P-~OCH2CH2 ~ OP ~ 0-3'-oligonucleotide O ONa ~ ONa O=P-O-Thymidine I

ONa 3~

-69~ 3 ~
Rça~g_nt Prepa ation A suspension of the polymeric particles (about 12-16% solids) was centrifuged (5000 rpm) and the resulting pellet was added to 2-(4-morpholino)-5 ethanesulfonic acid buffer (500 ~1, 0.1 molar, pH

6), and centrifuged again for. about 7 minutes, This procedure was repeated and finally the particles were resuspended in buffer (about 12% solids).
Four tubes containing the noted æuspension of particles were prepared by adding the suspension (380 ~1) noted above to the buffer (120 ~1). A
solution (100 ~1) of the activating agent (125 mg in 500 ~1 nanopure water) was added to each tube, and the resulting mixtures were mixed by manual end-over-end rotation for about 5 minutes at room temperature to provide activated polymeric particles.
A solution (2 OD) of each oligonucleotide was added to different tubes in the following volumes: 48.3 ~1 of #1, 37.7 ~1 of #2, 45.4 ~1 of #3 and 32.3 ~1 of #4. Each tube contained 600 ~1 of activated particles. The tubes were then rotated end-over-end at room temperature for about 18 hours to covalently bind the oligonucleotides to the activated particles.
The reaction mixtures were then centIifuged and the pellets resuspended in nanopure water (500 ~1) and centrifuged again. The treatment with the water and centrifugation was repeated twice more, and the f inal pellet was resuspended in nanopure water (600 ~1) to make a suspension of reagent (about 12%
solids). This suspension was diluted to about 1%
solids for use in an assay. The concentrations were determined spectrophotometrically by dilution and comparison with a standard dilution curve.

Example 16: Assay for Cvtomegaloviral DNA 2~l~3 Assay A for cytomegaloviral DNA was carried out using the reagents described above in Exa~ple 15 prepared USing 12% suspensions of polymeric particles. It was compared to Assay B carried out using the reagents of this i:nvention, but which were prepared using a prior art procedure using about 1-3%
suspensions of the polymeric particles.
Materials and Methods:
The target DNA to be detected was a 200 nucleotide segment of the late antigen (LA) region and a 265 nucleotide segment of the Major Immediate Early (MI~) region of the CMV Towne strain (ATCC VR
977).
The oligonucleotides used to detect the LA
region had the following sequences:
5'-GTCGAAGGCG GCTCGCTGGA AGCCGGTCGT~3' and 5'-GAACCGAGG CCGGCTCACC TCTATGTTGG-3'.
The primers used for LA region detection had the following sequences:
5' CACCACGCAG CGGCCCTTGA T~TTT-3' and 5'-GTCGCCTGCG CCAGGTGCTT CG-3'.
The oligonucleotides used to detect the MI~
region had the following sequences:
5'-GGTGTCACCC CCAGAGTCCC CTGTACCCGC-3' and 5'-GACACAGTGTCCTCCCGCTC CTCCTGAGCA-3'.
The primers for the MIE region had the following sequences:
5'-CAGCACCATC CTCCTCTTCC TCTGG-3' and 5'-GAGGCTATTG TAGCCTACAC TTTGG-3'.
The oligonucleotides were attached to the polymeric particles as described in Example 15 using about 12% suspensions of the particles to prepare the reagents (or probes) of this invention for Assay A.
The oligonucleotides were attached to the polymeric particles usin~ 1-3% suspensions of the particles to prepare reagents (or probes) ~or Assay B.

-71- 2 ~
The reagents for Assay A for the LA region were mixed (1 ~1 of a 0.5% suspension of each reagent), and spotted onto a defined area of the membrane (5 ~m LoProdyneTM microporous membrane, Pall Corp.) in the test wells of one set of SurecellTM test devices (Eastman Kodak Co.). The reagents ~or Assay A for the MIE region were similarly mixed and spotted onto a second defined area of the same membranes in the same devices. The applied reagents were allowed to dry for about 30 minutes at room temperature.
The rea~ents for Assay B (prepared using prior art procedures) for the LA region were mixed and spotted as described above in a second set of test devices. The reagents for Assay ~ for the MIE
region were mixed and spotted onto a second defined area of the same membranes.
A streptavidin-horseradish peroxidase conjugate was obtained from Zymed Labs (San Francisco?, and was diluted 1:8000 with a phosphate buffered saline solution containing casein (0.5~/O)~
3-(N-morpholino)propanesulfonic acid buffer (100 mmolar, pX 7.5) and preservative (0.01%). The final conjugate concentration was 156 ng/ml. The phosphate buffered saline solution contained sodium phosphate (25 mmolar, pH 7.3) and sodium chloride (75 mmolar).
A dye-providing composition was prepared containing 2-(4-hydroxy-3,5-dimethoxyphenol)-4,5-bis(4-methoxyphenyl)imidazole as ~ollows:
Solid leuco dye (to make a 0.1% solution) was dissolved in a solution of 20% poly(vinyl-pyrrolidone) in sodium phosphate buffer ~5 mmolar).
This solution was then added to a solution containing hydrogen peroxide (10 mmolar), 4'-hydroxyacetanilide electron agent (5 mmolar) and diethylenetriamine-pentaacetic acid chelating agent (10 ~molar) in --72- ~ it~
sodium phosphate buffer to product a final concen-tration of 1% poly(vinylpyrrolidone) and 0.005% leuco dye.
DNA polymerase, prepared according to the procedures described in EP-A-0 0258 017 (1 unit corresponds to 10 mmoles of dNTP incorporated into the primer extension product in 30 minutes at 37C), was obtained from Cetus Corp.
Assay:
To buf~er solutions containing tris(hydroxy-methyl)aminomethane buffer (lO mmolar, pH 8), potassium chloride ~50 mmola:r), magnesium chloride (10 mmolar) and gelatin (10 ~g) were added the primers described above (40 pmoles of each), dNTPs (1.5 molar of each) and the polymerase described above (7.5 units). In addition, the Towne strain targets described above were added. The total volume was 100 ~1.
~ach solution described above was placed into a polypropylene microfuge tube, primer extension products were formed, and amplification promoted using 33 consecutive thermal c~cles as follows:
66C rising to 92.5C 35 seconds 92.5OC 1 minute 92.5C lowering to 66C 45 seconds 66DC 1 minute After amplification, aliquots (5 ~1) of each mixture were added to a solution (95 ~1) of tris(hydroxymethyl)aminomethane buffer (10 mmolar, pH
8) containing potassium chloride (50 mmolar), magnesium chloride (10 mmolar) and gelatin (1 ~g/10 ml solution), heat denatured (5 minutes at 95C), then added to the test wells of the SurecellTM test devices described above (about 95 ~1 of each 3~ solution in individual ~ells).

The test devices were incubated for 5 minutes at 42C to hybridi2,e the amplified DNA target to the respective reagents (or probes) immobilized in the test wells. The hybridized products were washed with a buffered solution (250 ~1) containing phosphate buffer (10 mmolar, p~ 7,4), sodium chloride (150 mmolar), ethylenediaminetetraacetic acid (1 mmolar> and sodium decyl sul~ate ~1%) at 55~C.
The peroxidase-labeled avidin conjugate described above (50 ~1, 7.8 :ng) was added to each test well, and the devices were incubated at room temperature for 2 minutes. A second wash (250 ~1) was carried out using the buffered solution noted above. The leuco dye solution (100 ~1) was added to each test well followed by another incubation at room temperature for 2 minutes. The formation of dye was quenched by the addition of sodium azide (100 ~1 of 0.1% solution), and the resulting dye signals were evaluated on the membranes.
The amount of dye in each test device was visually evaluated a~ainst a color scale having values of 0 to 10 with 10 representin~ the highest dye density. The results are shown in Table X below for four separate tests for each of Assays A and B.
T A B L E X
Dye Densitv Readings LA Region MIE Region Assay A Assay B Assav A_ Assay B
Test 17.5 3 8 5.5 Test 27.25 2.5 8 5 Test 37 2.25 8 8 Test 47.25 2.5 8 5 Example 17: Comparative Example og_Rea~ents This example illustrates the preparation of several reagents of this invention and their -74- ~9~3~
comparison wlth reagents prepared us:ing polymers outside the scope of this invention.
The particles used to prepare the reagents were composed of the following copolymers:
Test A: Po~y(styrene-co-mono-m &
~-vinylbenzyl glutarate) (97.84:2.16 molar ratlo~, Test B: Poly(styrene-co-mono-~-vinylbenzyl glutarate--co-divinylbenzene) (97.01:2.16:0.83 molar ratio), Test C: Poly(styrene-co-mono-2-methacryloylo xyethyl glutarate) ~97.84:2.16 molar ratio), Test D: Poly(styrene-co-mono-2-methacryloylo xyethyl glutarate-co-divinyl-benzene) (97.01:2.16:0.83 molar ratio), Test E: Poly[styrene-co-monomethacryloyl-penta(oxyethylene) glutarate]
(98.7:1.3 molar ratio), Test F: Poly[styrene-co-monomethacryloyl-deca(oxyethylene) glutarate]
(99.2:0.8 molar ratio), Test G: Poly[styrene-~Q - mOnO - 2 - (m &
~-vinylbenzylthio)ethyl glutarate]
(98.3:1.7 molar ratio), Test H: Poly[styrene-co-mono 2-(~-vinyl-benzylthio)ethyl glutarate]
(98.25:1.75 molar ratio), Test I: Poly[styrene-co-3-(p-vinylbenzyl~
thio)propionic acid] (97.59: 2 . 41 molar ratio), Test J: Poly[styrene~co-mono-2-(4-vinyl-benzylthio)ethyl succinate]
(98.17:1.83 molar ratio), -75- 2 ~
Test K; Poly{styrene-co-4--[2-(carboxymetho xyaceto~y)ethylthiomethyl~-styrene} ~98.26:1.74 molar ratio), Test L: Poly(styrene co-mono-4-vinylbenzyl succinate) (97.71:2.29 molar ratio), Test M: Poly[styrene-co-mono-methacryloyl-penta(oxyethylene) phthalate]
(98.81:1.19 molar ratio), Control A Poly(styrene-çQ-acrylic acid) (95:5 molar ratio), and Control B Poly(styrene-co-3-acrylamido-3-methylbutanoic acid) (96.9:3.1 molar ratio).
Proteins were attached to the polymericparticles using l-(l-pyrrolidinylcarbonyl)pyridinium chloride as the activating agent. All particles were treated under the same conditions. The final dispersions comprised antibody (either tritiated bovine gamma globulin as in Example 1 above, or thyroxine antibodies as in Example 2 above) in an amount of 1.7 mg/m2 of polymer surface area in 2-(4-morpholino)ethanesulfonic acid buffer (0.1 molar, pH 5.5) and activating agent (1.5 mmole agent/g particles or 0.045 mmole).
These dispersions were prepared by adding suspensions of the particles (30 mg dry weight) to large microfuge tubes, and each was brought to a volume of 1.5 ml using the noted buffer. The resulting suspensions were centrifuged for 15 minutes at 13,000 spm and the supernatants discarded. Buffer (1 ml, 0.1 molar) was added to each tube, followed by addition of the activating agent (0.3 ml of a solution at 0.15 molar in buffer). The tubes were then capped and rotated end-over-end at room temperature for 10 minutes to provide activated~ ~ 3 polymeric particles.
A solution o~ the respective protein was added to respective tubes containing activated polymeric particles to provide a final concentration of 1,7 mg/m2 of polymer particles. Tritiated bovine gamma globulin was ,added to certain tubes while thyroxine monoclonal antibodies (Cambridge/Ventrex) were added to other tubes.
Buffer was then added to all tubes to achieve a final volume of 1.5 ml. The tubles were rotated end-over-end at room temperature for 24 hours to react the protein with the activated particleæ.
Reaction of antibodies with particles was quenched by the addition of bovine serum albumin (250 ~1, 100 mg protein/ml) to each tube. The tubes were then rotated again for an additional 16 hours at room temperature.
The reagents having attached 3H bovine gamma globulin were treated as described in Example 1 to determine the total amount of protein attached as well as the amount covalently bound.
The reagents having thyroxine antibodies attached were washed and the relative amounts of active antibody in the resulting dispersions were determined as described in Example 2.
The results are summarized in Table XI
below. The results indicate that the tritiated bovine gamma globulin was covalently bound very well in most of the reagents of this invention. Reagents of Tests E.and F did not covaiently bind the protein as well as the other reagents. However, when an aromatic carboxy functional group was added to the end of the linking group (as for the reagent in Test M), the amount of covalently bound protein was increased substantially.

2 ~

The reagents having bound antibodies showed 3-20 times more activity than the reagents in the Controls, as indicated in the last column of Table XI.
T A B L E XI
Theoretical Thyroxine Covalent: Binding Sites where Total % % Covalent Total 50% of Label is _est Bound Bound Ratio ~a~ ~nmolar) A 97 96 O.g9 2.4 B 96 95 0.99 3.2 C 96 96 :L.0 2.6 D 95 95 :L.0 3.2 E 52 48 0.94 2.4 F 31 23 0.75 3.5 G 97 96 0.99 3.5 H 96 95 1.. O 2.4 I 97 95 0.99 3.5 J 96 95 0.99 2.9 K 94 94 1.00 1.8 L 96 95 0.99 3.5 M B9 87 0.98 2.1 Control A 94 90 0.97 12 Control ~5 B 96 93 0,97 42 Example 18: ImmunQassav for Thvroxine Usin~
Drv Analvtical Element This example illustrates the preparation of a dry analytical element of this invention and its use in a competitive immunoassay to determine the hormone thyroxine in a liquid sample. Two reagents of this invention are compared to a Control reagent in the assays.
Two reagents prepared accoxding to the description in Example 2 were used in this assay. A
Control reagent was prepared using the procedures described in ~P-A-0 280 556. The activating agent used was l~ pyrrolidinyl- carbonyl)pyridinium chloride. The reagents were incorporated into standard barium sulfate spreading layers of the elements at two levels: either 25 mg polymer/m2 of layer surface, or 50 mg polyimer/m2 of layer 6urface.
The copolymers used to prepare the reagents were:
Test A Poly(styrene-co-mono-_ ~-vinylbenzyl glutarate) (97.84:2.16 molar ratio), Test B Poly(styrene-co-mono-2~methacryloylo xyethyl glutarate) (97.84:2.16 molar ratio), and Control A core/shell polymer particle having a core of poly(styrene-co-ethylene dimethacrylate) (99:1 molar ratio~
and a shell of poly[styrene-co~m &
~-(2-chloroethylsulfonylmethyl)-styrene-~-ethylene dimethacrylate] (94.S:4.5:1 molar ratio).

2 ~ e3 The structure of the element used in the assay was as follows:

Coverage (glm2~
Spreading Barium sulfate 108.6 Layer Reagent with thyroxine antibodies 0.0~5 or 0.05 Cellulose acetate 8.6 EstaneTM polyurethane 1.08 (B.F. Goodric.h) TritonTM X-405 surfactant 2.1 (Rohm and Haas) Subbing Poly(N-isopropyl-Layer 2 acrylamide) 13.1 Gelatin Triethanolamine (pH 7.5) 0.15 Subbing Bovine serum albumin 0.05 Layer 1 TritonTMX-100 surfactant ~Rohm & Haas) 0.01 Magnesium chloride 0.02 Zinc chloride 0.001 Hardened gelatin 10 Gelatin Triethanolamine (pH 7.5) 0.75 Layer TritonTMX-100 surfactant (Rohm & Haas) 0.02 Poly(ethylene térephthalat~
Support A series of thyroxine standard solutions varying in concentration from ~0 5 to 10 10 molar ? ~ ~ 9 were prepared in the buffer described below fxom a 1 x 10 3 molar thyroxine stock solution in dimethyl sulfoxide. The buffer consisted of phosphate buffered saline (pH 7.4), bovine serum albumin (1%) 5 and 8-anilino-1-naphthaleneslllfonic acid (~.7 x 10 4 molar). A solution of alkaline phosphatase labeled thyroxine was prepared at a concentration of 2.0 x 10 8 molar. The labeled analogs were prepared by the method descr:ibed by Ito et al, Clin.Ch~, 30(10), pp. 1682--1685 (1984>. The thyroxine standards and the :labeled analog solution were mixed 1:1, so that the final concentration of labeled analog was 1.0 x 10 8 molar.
Ali~uots (10 ~1) of this mixture were spotted onto the spreading layer of the element.
After 5 minutes incubation at 37C, a wash solution (10 ~1) containing ~-nitrophenyl phosphate (15 mmolar) was added to the element to remove uncomplexed labeled analog from any complex formed in the spreading layer. After a one minute incubation, the change in reflection density (~DR) was measured for 30 seconds in the center of the element at 37C and 400 nm using a standard spectrophotometerO
The results of the assays for the two ~5 thyroxine concentrations are provided in Table XII
below. These results show that the thyroxine antibody immobilized on two reagents of this invention in the element provides improved results over the Control reagent. More specifically, the reagents of this invention exhibit a "higher reaction" rate where the thyroxine level is low.
Thus, the reagents exhibit high binding reactivity towards the labeled analog and provide a greater working range for the assay. Working range is defined here as the difference in rates at 10 10 and 10 5 molar thyroxine.

-81- 2 ~
T A B L E XII
Antibody Rate (AD /min.) R
Coverage Working Test (g/m2) 10 lmolar 10 5molar _ Ran~e 5A 0.05 0.072 0.0050.067 ~ 0.05 0.090 0.0050.085 Control 0.05 0.042 0.0010.041 A 0.10 0.103 0.0100.093 B 0.10 0.112 0.0100.102 10Control 0.10 0.068 0.0020.066 Example 19: Assay for Thyroxine in Drv Element This example illustrates the practice of this invention to detect thyroxlne in a dry analytical element using a competitive binding assay and a horseradish peroxidase-labeled conjugate.
Two thyroxine antibodies were used: (a) obtained from BiosPacific (clone 035-A2206A), and (b) obtained from Beckman Instruments (#684823). They were covalently attache~ to particles of poly-[styrene-co-3-(p-vinylbenzylthio)propionic acid]
(97.59:2.41 molar ratio) using the procedure described in Example 2 and l-(l-pyrrolidinyl-carbonyl)pyridinium chloride as an activating agent.
The resulting reagents ~ere incorporated into a spreading layer o~ the analytical element illustrated 3~

below.
Coverage _ _ _ _ _~gLm Reagent (1.4 ~m particles) 0.15 Particles of polyCm= & ~
~inyltoluene (64:36 >-CQ-methacrylic acid)(98:2 weight ratio) (30 ~m) 129 3,4-Bis(4-dimethylamino-phenyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)imidazole 0.2 3-(4-morpholino)propanesul-Spreading fonic acid buffer 0.2 15Layer ZonylT~FSN surfactant (DuPont) 0.054 Xanthan gum (KelzanTM from Kelco) 0.065 5,5-Dimethyl-1,3-cyclohexane-dione 0.050 Poly(methyl acrylate-cQ-sodium 2-acrylamido-2-methyl pro-panesulfonate-co-2-aceto-acetoxyethyl methacrylate) (90:4:6 weight ratio) 2.58 Hardened gelatin 10 Gelatin Potassium phosphate 0.68 Layer TritonTMX-100 surfactant ~Rohm ~ Haas) 0.02 4'-hydroxyacetanilide 0.15 Poly(ethylene Térephthalats) Suppor~

A series of thyroxine standard solutionæ
were prepared in the buffer described below from a -83~ 9 10 3 molar stock solution (in dimethyl sulfo~ide) to have concentration varying from 10 5 to 10 10 molar. The buffer consisted of 3-(4-morpholino)-propanesulfonic acid bu~fer (0.2 molar, pH 7), 4-hydroxyacetanilide (0.01 molar), 8-anilino-naphthalene-l-sulfonic acid 8.7 x 10 4 molar) and bovine gamma globulin (0.1%) A horseradish peroxidase-labeled thyroxine analog solution was prepared at a concentration of 2 x 10 9 molar. The analog was prepared using the method described by Kunst et al, Clin.Chem., 34(9), pp. 1830-1833 (1988).
The analog and standard solutions were mixed 1:1 so that the final concentration of the analog was 10 9 molar. A Control solution containing no thyroxine was also tested.
Aliquots (10 ~1) of the mixtures were spotted onto the spreading layer of the element.
After incubation at 37C for 5 minutes, a wash solution containing hydrogen peroxide (10 ~1) was added to wash uncomplexed analog from the complex in the center of the element. After incubation for 40 seconds at room temperature, the change in reflection density ~DR) was measured for 30 seconds in the center of the element at 37C and 680 nm using a standard spectrophotometer. The Williams~Clapper transform (J.Opt.Soc.Am., 43, 595, 1953) was used to convert the reflection densities to transmittance values (~DT). The results are shown in Table XIII below. They show substantial changes in the rate in the desired thyroxine concentration range using the two reagents of this invention.

3~

3~

T A B L E XIII
Thyroxine Rate (~DT/Min.) Concentration (Molar2 __ Reagent (a~ Reagent (b~
Control 0.0877 0,0914 lO_lo 0.089~ 0.0942 10-9 0.0888 0.09~1 ~8 0.0802 0.0867 10-7 0.0398 0.0396 10-6 0.0226 0.0245 10-5 0.01~7 The invention has been described in detail with particular reference to preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention. Moreover, all patents, patent applications (published or unpublished, foreign or domestic), literature references or other publications noted above are incorporated herein by reference for any disclosure pertinent to -the practice of this invention.

~5

Claims (53)

1. A biologically active reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a biologically active substance covalently attached to said particle through said reactive carboxy group or salt thereof.

2. The reagent of claim 1 wherein said biologically active substance is selected from the group consisting of amines, enzymes, amino acids, peptides, polypeptides, proteins, lipoproteins, glycoproteins, hormones, drugs, steroids, vitamins, polysaccharides, glycolipids, alkaloids, microorganisms, viruses, protozoa, fungi, parasites, rickettsia, molds, blood components, tissue and organ components, pharmaceuticals, haptens, lectins, toxins, nucleic acids, antigenic materials, biotin or derivatives thereof and components thereof.

3. The reagent of claim 2 wherein said biologically active substance is an immunoreactive species.

4. The reagent of claim 3 wherein said biologically active substance is an antibody.

5. The reagent of claim 4 wherein said biologically active substance is an antibody directed to Streptococcus A, a microorganism associated with periodontal disease, carbamazepine, thyroxine, human chorionic gonadotropin, phenobarbital, phenytoin or digoxin.

6. The reagent of claim 3 wherein said biologically active substance is an antibody directed to human chorionic gonadotropin, phenobarbital, phenytoin, digoxin or thyroxine.

7. The reagent of claim 2 wherein said biologically active substance is an oligonucleotide.

8. The reagent of claim 7 wherein said oligonucleotide is complementary to a nucleic acid sequence of HIV-I DNA, .beta.-globin DNA or cytomegalovirus DNA.

9. The reagent of claim 1 wherein said copolymer has recurring units derived from about 85 to about 99.5 mole percent of monomer (a), from about 0.5 to about 15 mole percent of monomer (b), and from O to about 10 mole percent of monomer (c).

10. The reagent of claim 9 wherein R is hydrogen or methyl, M is hydrogen or an alkali metal ion, and L comprises two or more alkylene or arylenealkylene groups which are connected or terminated with an oxy, thio, imino (-NR1-), carbonyloxy (-COO-), carbonylimino (-CONR1-), ureylene (-NR1CONR1-) or sulfonylimino (-SO2NR1-) group, wherein each R1 is independently hydrogen, alkyl having 1 to 10 carbon atoms, cycloalkyl having 4 to 10 carbon atoms or aryl having 6 to 14 carbon atoms.

11. The reagent of claim 10 wherein L is p-phenylenemethyleneoxycarbonyltrimethylene, carbonyloxyethyleneoxycarbonyltrimethylene, carbonyloxyethyleneureylenepentamethylene, carbonylpenta(oxyethylene)oxycarbonyltrimethylene, carbonyldeca(oxyethylene)oxycarbonyltrimethylene, p-phenylenemethylenethioethyleneoxycarbonyltri-methylene, carbonyloxyethyleneiminocarbonyl-trimethylene, carbonyloxytetramethyleneoxycarbonyl-tetramethylene, p-phenylenemethyleneiminocarbonyl-trimethylene, p-phenylenemethyleneiminocarbonyl-trimethylene, p-phenylene(methyl)iminoethylene-oxycarbonyltri-methylene, p-phenylenemethylenethio-ethylene, p-phenylenemethylenethioethyleneimino-carbonylmethylene-oxymethylene, p-phenylenemethylene-thioethyleneimino-carbonylmethylenethiomethylene, p-phenylene-methylenethioethyleneiminocarbonyltri-methylene, phenylenemethylenethio-1-carboxyethylene, phenylenemethylenethiophenylene, phenylenemethylene-thioethyleneoxyethylenethiomethylene-oxycarbonyl-ethylene, phenylenemethyleneoxyphenylenemethylene-thioethylene, phenylenemethylenethioethyleneoxy-ethylenethioethyleneoxycarbonylethylene, phenylene-methyleneoxyphenylenemethylenethiophenylenemethylene-thiotrimethylene and phenylenemethylenethioethylene-oxyethylenethioethyleneoxycarbonylphenylene.

12. The reagent of claim 1 wherein monomer (b) is selected from the group consisting of:
mono-m & p-vinylbenzyl glutarate, mono-p-vinylbenzyl glutarate, mono-2-methacryloyloxyethyl glutarate, 2-(4-carboxybutyramido)ethyl methacrylate, 2-[N'-(5-carboxypentyl)ureido]ethyl methacrylate, mono-methacryloylpenta(oxyethylene) glutarate, mono-(4-acryloyloxybutyl) glutarate, 4-(4-carboxy-butyramido)styrene, mono-methacryloyldeca-(oxyethylene) glutarate, mono-2-(p-vinylbenzylthio)-ethyl glutarate, mono-2-(m & p-vinylbenzylthio)ethyl glutarate, 4-(4-carboxybutyramidomethyl)styrene, mono-2-[N-methyl-N-(4-vinylbenzyl)amino]ethyl glutarate, 3-(p-vinylbenzylthio)propionic acid, 4-[2-(4-carboxybutyramido)ethylthiomethyl]styrene, 4-[2-(carboxymethoxyacetamido)ethylthiomethyl]styrene, 4-[2-(carboxymethylthioacetamido)ethylthiomethyl]-styrene, mono-2-(4-vinylbenzylthio)ethyl succinate, 4-[2-(carboxymethoxyacetoxy)ethylthiomethyl]styrene, mono-4-vinylbenzyl succinate, 2-(4-vinylbenzylthio)-succinic acid, 2-(4-vinylbenzylthio)benzoic acid, mono-2-[2-(4-vinylbenzylthio)ethoxy]ethylthiomethyl malonate, mono-methacryloylpenta(oxyethylene) phthalate, mono-methacryloyldeca(oxyethylene) phthalate, mono-2-{2-[2-(4-vinylbenzylthio) ethoxy]ethylthio}ethyl succinate, mono-2-{2-[2-(4-vinylbenzylthio)ethoxy]ethylthio}ethyl phthalate, 3-[4-(4-vinylbenzyloxy)benzylthio]-propionic acid and 4-{4-[4-(4-vinylbenzyl-oxy)benzylthio]benzylthio}butyric acid.

13. The reagent of claim 12 wherein monomer (b) is 3-(p-vinylbenzylthio)propionic acid.

14. An analytical element comprising a fluid-permeable substrate having one or more reaction zones therein, and containing in at least one of said zones, a biologically active reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:

(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a biologically active substance covalently attached to said particle through said reactive carboxy group or salt thereof.

15. The element of claim 14 wherein said biologically active substance is an antibody.

16. The element of claim 15 wherein said biologically active substance is an antibody to carbamazepine, thyroxine, phenobarbital, phenytoin or digoxin.

17. The element of claim 14 wherein said copolymer has recurring units derived from about 85 to about 99.5 mole percent of monomer (a), from about 0.5 to about 15 mole percent of monomer (b), and from 0 to about 10 mole percent of monomer (c), and wherein R is hydrogen or methyl, M is hydrogen or an alkali metal ion, and L comprises one or more alkylene or arylenealkylene groups which are connected or terminated with an oxy, thio, imino (-NR1-), carbonyloxy (-COO-), carbonylimino (-CONR1-), ureylene (-NR1CONR1-) or sulfonylimino (-SO2NR1-) group, wherein each R1 is independently hydrogen, alkyl having 1 to 10 carbon atoms, cycloalkyl having 4 to 10 carbon atoms, or aryl having 6 to 14 carbon atoms.

18. An analytical element comprising a nonporous support, having imposed thereon, in order and in fluid contact, a reagent layer containing one or more reagents for providing a detectable signal in the assay, a water-soluble layer containing a detectably labeled analog of the ligand of interest, and a porous spreading layer containing a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or mole additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a receptor for said ligand of interest covalently attached to said particle through said reactive carboxy group or salt thereof.

19. The element of claim 18 wherein said receptor is an antibody directed to phenobarbital, phenytoin, digoxin or thyroxine.

20. A method for the determination of a specific binding ligand comprising:
A. forming a water-insoluble specific binding complex of a specific binding ligand of interest, or a receptor therefor, with a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a biologically active substance covalently attached to said particle through said reactive carboxy group or salt thereof, said substance being specifically reactive with either said ligand or a receptor therefor, and B. detecting the presence of said complex as an indication of the presence or amount of said ligand in said specimen.

21. The method of claim 20 wherein said ligand is a protein, carbohydrate, hapten, hormone or drug, and said receptor is an antibody for said protein, carbohydrate, hapten, hormone or drug.

22. The method of claim 20 wherein said ligand is biotin or a derivative thereof, and said receptor is avidin or a derivative thereof.

23. The method of claim 20 wherein said ligand is a single-stranded nucleic acid, and said receptor is a second single-stranded oligonucleotide which is substantially complementary to said nucleic acid.

24. A competitive binding assay for the determination of a specific binding ligand comprising:
A. contacting a specimen suspected of containing a water-soluble specific binding ligand with a water-soluble receptor therefor, and with a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:

(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) molecules of said ligand covalently attached to said particle through said reactive carboxy group or salt thereof, to form specific binding complex (a) between said receptor and said ligand, and specific binding complex (b) between said receptor and said water-insoluble reagent, and B. after separating said complexes (a) and (b), detecting the presence of either complex as an indication of the presence or amount of said ligand in said specimen.

25. A method for the determination of an immunological species comprising:

A. contacting a specimen suspected of containing an immunological species with a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a receptor for said species covalently attached to said particle through said reactive carboxy group or salt thereof, to form a water-insoluble immunological complex of said species with said receptor, and B. after separating uncomplexed materials from said complex, detecting the presence of said complex as an indicator of the presence or amount of said immunological species in said specimen.

26. The method of claim 25 for the determination of an antigenic material wherein said receptor is an antibody for said antigenic material.

27. The method of claim 25 for the determination of an antibody wherein said receptor is an antigenic material specific for said antibody.

28. The method of claim 25 for the determination of a first antibody wherein said receptor is a second antibody directed to said first antibody.

29. The method of claim 25 wherein said uncomplexed materials are separated from said immunological complex by filtration with a microporous membrane.

30. The method of claim 25 wherein said reagent is immobilized on a microporous filtration membrane.

31. The method of claim 25 wherein, either prior to, simultaneously with or subsequently to the formation of said water-insoluble immunological complex, said immunological species is reacted with a water-soluble specific binding component specifically reactive therefor.

32. The method of claim 31 wherein said specific binding component is labeled for detection.

33. The method of claim 31 wherein said label is an enzyme or radioisotope.

34. The method of claim 26 for the determination of human chorionic gonadotropin, Streptococcus A or a microorganism associated with periodontal disease.

35. An assay for the determination of a specific binding ligand comprising:
detecting the presence or amount of a water-insoluble specific binding complex formed between a ligand of interest and a receptor therefor, the receptor provided as a component of a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:

CH2=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) the receptor for said ligand being covalently attached to said particle through said reactive carboxy group or salt thereof.

36. An immunoassay employing antibodies or antigens for detecting the presence or amount of a ligand in a specimen, said immunoassay comprising addition of an immunoreactant which is specifically reactive with said ligand or with a receptor therefor, said immunoreactant being a component of a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:

CH2=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) the immunoreactant being covalently attached to said particle through said reactive carboxy group or salt thereof.

37. An analytical separation method comprising:
A. passing a specimen containing a mixture of biologically active substances over an affinity chromatography reagent comprising:

( (I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:

CH=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a specific binding substance covalently attached to said particle through said reactive carboxy group, said specific binding substance being specific to one or more predetermined biologically active substances in said specimen mixture of biologically active substances to form a complex of said reagent with said predetermined substances, and B. collecting either the one or more complexed predetermined substances or one or more substances remaining in the eluent.

38. A method for the detection of a nucleic acid comprising:
A. forming a water-insoluble hybridization product between a nucleic acid of interest, with a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:

CH=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) an oligonucleotide covalently attached to said particle through said reactive carboxy group or salt thereof, said oligonucleotide being substantially complementary to said nucleic acid of interest, and B. detecting the presence of said hybridization product as an indication of the presence or amount of said nucleic acid of interest.

39. The method of claim 38 wherein said nucleic acid of interest is amplified using polymerase chain reaction prior to contact with said reagent.

40. The method of claim 38 for the detection of HIV-I DNA, cytomegaloviral DNA or .beta.-globin DNA.

41. The method of claim 39 carried out using a set of primers, one of which is biotinylated, and detection of the nucleic acid of interest is accomplished using an enzyme-avidin conjugate.

42. The method of claim 38 wherein said hybridization product is captured on a microporous membrane.

43. The method of claim 38 wherein said hybridization product is captured on a nonporous solid substrate.

44. A kit for a hybridization assay for a nucleic acid of interest comprising:
a. a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:
CH=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) an oligonucleotide covalently attached to said particle through said reactive carboxy group or salt thereof, said oligonucleotide being substantially complementary to a nucleic acid of interest, and b. one or more additional reagents, solutions or equipment needed for said assay.

45. A kit for a specific binding assay for the determination of a ligand of interest comprising:
a. a reagent comprising:
(I) a water-insoluble particle composed of, at least on its surface, a copolymer having recurring units derived from:
(a) from about 60 to about 99.8 mole percent of one or more ethylenically unsaturated polymerizable oleophilic monomers which provide hydrophobicity to said copolymer, (b) from about 0.2 to about 40 mole percent of one or more ethylenically unsaturated polymerizable monomers having a reactive carboxy group, or salt thereof, and represented by the structure:

CH=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain, and (c) from 0 to about 15 mole percent of one or more additional ethylenically unsaturated polymerizable monomers other than those identified in categories (a) and (b) above, and (II) a biologically active substance covalently attached to said particle through said reactive carboxy group or salt thereof, said substance being specifically reactive with either said ligand of interest or a receptor therefor, and b. one or more additional reagents, solutions or articles needed for said assay.

46. A method for the preparation of a nucleic acid reagent comprising:
A. contacting (1) an aqueous suspension of carboxylated polymeric particles having an average size of from about 0.01 to about 20µm, said particles being present at at least about 5% solids with (2) an activating agent to produce reactive intermediate polymer particles having intermediate reactive groups, and B. contacting the reactive intermediate polymer particles produced in step A with an oligonucleotide having a reactive amine or sulfhydryl groups which reacts with said intermediate reactive groups to form a covalent linkage between said particles and said oligonucleotide.

47. The method of claim 46 wherein said activating agent is a carbamoylonium compound.

48. The method of claim 47 wherein said activating agent is a water-soluble carbodiimide.

49. The method of claim 46 wherein said carboxylated polymeric particles are represented by the structure:
wherein A represents recurring units derived from one or more ethylenically unsaturated polymerizable mono-mers containing carboxyl groups or salts or precursors of said groups, B represents recurring units derived from one or more ethylenically un-saturated polymerizable monomers, and x is from about 0.1 to about 70 mole percent.

50. The method of claim 49 wherein B is derived from styrene or a styrene derivative, an acrylic or methacrylic acid ester, or acrylonitrile, x is from about 1 to about 20 mole percent, and A is derived from an ethylenically unsaturated polymerizable monomer represented by the structure:

CH=?-L-?-O-M

wherein R is hydrogen, halo or alkyl of 1 to 3 carbon atoms, M is hydrogen, an alkali metal ion or an ammonium ion and L is an organic linking group having from 8 to 50 atoms selected from the group consisting of carbon, nitrogen, oxygen and sulfur atoms in the linking chain.

51. The method of claim 50 wherein R is hydrogen or methyl, M is hydrogen or an alkali metal ion, and L comprises two or more alkylene or arylenealkylene groups which are connected or terminated with an oxy, thio, imino (-NR-) carbonyloxy (-COO-), carbonylimino (-CONR1-), ureylene (-NR1CONR1-) or sulfonylimino (-SO2NR1-) group, wherein each R1 is independently hydrogen, alkyl having 1 to 10 carbon atoms, cycloalkyl having 4 to 10 carbon atoms or aryl having 6 to 14 carbon atoms.

52. The method of claim 46 wherein said oligonucleotide is complementary to .beta.-globin DNA, HIV-I DNA or cytomegaloviral DNA or a sequence thereof.

53. The method of claim 46 wherein said polymeric particles are present in said suspension at from about 10% to about 25% solids.