Method of preparing biologically active reagents from succinimide-containing polymers, analytical element and methods of use

Biologically active reagents are prepared from particles of copolymers having highly active succinimid groups. The reagents are prepared by covalently attaching biologically active substances, for example antibodies, to the particles, directly or indirectly through amide groups by displacement of highly active succinimid 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 such as affinity chromatography.

RELATED APPLICATION 
Reference is made to copending and commonly assigned U.S. Ser. No. 
07/646,132, filed on even date herewith by Sutton, Ponticello, Danielson 
and Oenick, and entitled "Succinimide Containing Monomers and Polymers and 
Latices Prepared from Same", now U.S. Pat. No. 5,200,462. 
FIELD OF THE INVENTION 
The present invention relates to a method of preparing biologically active 
reagents which uses polymeric particles. It also relates to analytical 
elements containing such biologically active reagents, and to immunoassays 
and specific binding analytical methods using them. Further, it relates to 
an analytical purification method using the reagents. This invention can 
be used for various clinical, diagnostic, medical and research purposes. 
BACKGROUND OF THE INVENTION 
There is 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. 
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 have 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 
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 from a mixture of such materials. 
U.S. Pat. No. 4,278,651 (issued Jul. 14, 1981 to Hales) relates to a 
supported receptor for use in an assay for a ligand in which the solid 
support contains a water insoluble polymer having available at least one 
active functional group which is either carboxyl, isothiocyanate, 
N-hydroxysuccinimid, imidazolide, bromoacetyl, maleimide or 
diazomethylene. The receptor having been covalently linked to the support 
through the active functional group. Generally, the support is a large 
core-shell particle having an outer porous coating as the shell which also 
has the necessary functional groups. The core of the particle provides 
structural integrity for the porous shell materials. 
It would be expected that porous particles would provide greater surface 
area over non-porous particles. This, in practice, is not the case. 
Depending on the size of the pores, porous particles are relatively 
inefficient for use with large molecules of biological interest. For 
example, a useful ligand may be attached to a porous bead in one of the 
pores. The biological species which it is desired to eliminate or separate 
from the liquid stream may be so large as not to be able to get down into 
the pores and reach the ligand. Conversely, immobilization of a large 
affinity ligand can only take advantage of a small portion of the total 
surface area since the ligand itself cannot penetrate the porous well. 
Thus, the efficiency of binding is diminished and the apparent surface 
area advantage of such porous particles becomes illusory. 
Acrylic acid-based photopolymerizable compositions have been prepared which 
are capable of binding bioactive substances after being photopolymerized, 
as described in U.S. Pat. No. 4,451,569 (issued May 29, 1984 to Schneider 
et al). These compositions may be applied as a coating on a carrier 
substrate, photopolymerized and a bioactive substance fixed thereto. The 
composition contains acrylic acid, a photoinitiator, a photopolymerization 
activator and adhesion promoter, and a copolymerizable olefinic monomer 
which contains a active functional group capable of binding bioactive 
substances. The olefinic monomer is preferably 
N-hydroxysuccinimidacrylate, N-hydroxysuccinimid amidocaproate, 
epoxypropyl acrylate or 2-isocyanato-ethyl acrylate. 
Also, biologically active substances have thus been immobilized to 
advantage on particulate substrates such as polymeric particles, animal 
and human erythrocytes, bacterial cells and other materials known in the 
art. In some cases, the particulate substrates are fashioned or chemically 
treated to provide active groups on their outer surfaces for appropriate 
reaction with the biological substance. If the particulate substrate is a 
polymeric material, it often can be prepared from monomers having the 
appropriate active groups. 
For example, carboxylated latex particles have been used to prepare 
diagnostic reagents, as noted in U.S. Pat. No. 4,181,636 (issued Jan. 1, 
1980 to Fischer). 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 U.S. Pat. No. 3,857,931 (issued Dec. 31, 1974 
to Hager), U.S. Pat. No. 4,138,383 (issued Feb. 6, 1979 to Rembaum et al) 
and U.S. Pat. No. 4,264,766 (issued Apr. 28, 1981 to Fischer). 
For example. U.S. Pat. No. 4,710,525 (issued Dec. 1, 1987 to Kraemer) 
relates to certain polymer particles dispersible to form a latex, to 
latices of such polymer particles, and to methods for immobilizing (i.e., 
bonding or fixing) a biologically active substance on such particles. 
These particles have a core-shell construction and comprise groups in the 
shell region which are suitable for covalent fixation thereto of a 
biologically active substance. The shell construction is also hydrophilic 
and crosslinked. The crosslinking is necessary to prevent dissolution of 
the very hydrophilic shell polymer. The crosslinked hydrophilic matrix of 
Kraemer has the disadvantage of being nonabsorptive towards antibodies and 
other proteins to be adsorbed or immobilized on the surface of the 
antibody or protein. A material that adsorbs to a hydrophilic surface can 
be more easily displaced than if it were adsorbed to a more hydrophobic 
surface. 
Two known monomers, N-acryloyloxysuccinimid and 
N-(6-methacrylamidohexanoyloxy)succinimid, have been polymerized to form 
polymers. These monomers are generally water-insoluble, but are difficult 
to copolymerize with oleophilic monomers by emulsion polymerization in 
water and are not readily polymerized to form monodisperse particles. 
Notwithstanding the current status of the arts of medical practice and 
analytical and diagnostic procedures, there is a need in the industry for 
a method of preparing biologically active reagents having water-insoluble, 
non-porous particles of a non-crosslinked copolymer useful in this 
invention. 
SUMMARY OF THE INVENTION 
The problems noted above are overcome with a method of preparing 
biologically active reagent comprising the step of reacting: 
(I) a water-insoluble, nonporous particle of a noncrosslinked copolymer 
having recurring units derived from: 
(a) from 0 to about 99.9 mole percent of one or more ethylenically 
unsaturated polymerizable oleophilic monomers which provide hydrophobicity 
to a copolymer, provided that none of the monomers are crosslinking 
monomers, 
(b) from about 0.1 to 100 mole percent of one or more ethylenically 
unsaturated polymerizable monomers having a succinimidoxycarbonyl group, 
and 
(c) from 0 to about 10 mole percent of one or more other hydrophilic 
ethylenically unsaturated polymerizable monomers, with 
(II) a biologically active substance having been covalently attached to a 
particle through an amide group by displacement of the succinimidoxy 
portion of said succinimidoxycarbonyl group. 
This invention also provides an analytical element comprising a 
fluid-permeable 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, this invention provides 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 in response to an enzyme, 
a water-soluble layer containing an analog of a ligand labeled with the 
enzyme, and 
a porous spreading layer containing a biologically active reagent 
comprising: 
(I) the particle as described above, and 
(II) a receptor for the ligand having been covalently attached to the 
particle through an amide group by displacement of the succinimidoxy 
portion of a succinimidoxycarbonyl group. 
Further, this invention provides a method for the determination of a 
specific binding ligand comprising: 
A. contacting a specimen suspected of containing a water-soluble specific 
binding ligand with the reagent as described above, 
to form a water-insoluble specific binding complex of the ligand with the 
receptor, and 
B. detecting the presence of the complex as an indication of the presence 
or absence of the ligand in the specimen. 
Even further, this invention provides a method for the determination of a 
specific binding ligand comprising: 
A. contacting a specimen suspected of containing a water-soluble specific 
binding ligand with the reagent as described above, 
(I) the particle as described above, and 
(II) molecules of the ligand having been covalently attached to the 
particle through an amide group by displacement of the succinimidoxy 
portion of a succinimidoxycarbonyl group, 
B. prior to, simultaneously with or subsequent to the contact in step A, 
contacting the specimen with a receptor for the ligand to form a 
water-soluble specific binding complex of the receptor with the 
water-soluble ligand and a water-insoluble specific binding complex of the 
receptor with the water-insoluble ligand, 
C. separating the water-insoluble complex from water-soluble materials, and 
D. detecting the presence of the water-insoluble complex as an indication 
of the presence or absence of the ligand in the specimen. 
Also, this invention provides 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) the particle as described above, and 
(II) a receptor for the species having been covalently attached to the 
particle through an amide group by displacement of the succinimidoxy 
portion of a succinimidoxycarbonyl group, 
to form a water-insoluble immunological complex of the species with the 
receptor, 
B. removing water-soluble materials from the complex, and 
C. detecting the presence of the complex as an indicator of the presence or 
absence of the immunological species in the specimen. 
This invention further provides an analytical purification method 
comprising: 
A. passing a specimen containing a mixture of biologically active 
substances over an affinity chromatography reagent comprising: 
(I) the particle as described above, and 
(II) a specific binding substance having been covalently attached to the 
particle through an amide group by displacement of the succinimidoxy 
portion of a succinimidoxycarbonyl group, the specific binding substance 
being specific to one or more predetermined biologically active substances 
in the specimen mixture of biologically active substances, and 
B. collecting the one or more predetermined biologically active substances 
on the reagent. 
The present invention provides reagents which are useful in a variety of 
analytical, diagnostic and purification methods. 
The advantages of the present invention are that the use of certain 
copolymers having a succinimid group are more easily and more completely 
incorporated into water-insoluble latex particles, and thereby more easily 
facilitate attachment of proteins or other biological compounds. Moreover, 
the copolymers useful in this invention have the following advantages: (1) 
water-insolubility, (2) they do not swell in water, (3) they are 
colloidally stable to the biological chemistries of immobilization, (4) 
they are surfactant-free, (5) they are polymer protective colloid-free, 
(6) they are non-porous, (7) they are non-crosslinking, and (8) they 
monodisperse. 
Detailed Description of the Invention 
The copolymers useful in the method of this invention are described in 
detail in U.S. Ser. No. 07/646,132 of Sutton, et al (noted above). The 
following discussion is provided as a summary of these copolymers. 
The copolymers have essential recurring units derived from: 
(a) from 0 to about 99.9 preferably from 80.0 to about 99.9, more 
preferably from 90.0 to about 99.9, and most preferably from 95.0 to about 
99.9, mole percent of one or more ethylenically unsaturated polymerizable 
oleophilic monomers which provide hydrophobicity to said copolymer, 
provided that none of the monomers are crosslinking monomers, that is, 
monomers having 2 or more addition polymerizable vinyl groups capable of 
polymerizing to form a 3-dimensional, crosslinked polymer network, 
(b) from about 0.1 to 100, preferably from 0.1 to about 20 to, more 
preferably from 0.1 to about 10, and most preferably from 0.1 to about 
5.0, mole percent of one or more ethylenically unsaturated polymerizable 
monomers having a succinimidoxycarbonyl group, and 
(c) from 0 to about 10, preferably from 0 to about 5, and more preferably 
from 0 to about 3, mole percent of one or more other non-crosslinking 
ethylenically unsaturated polymerizable monomers, such as ionic or polar 
hydrophilic monomers. 
Preferably, the copolymer comprises monomer (b), as described above, which 
is represented by the structure: 
##STR1## 
wherein: 
R is hydrogen, alkyl of 1 to 3 carbon atoms or halo, 
L is a linking group having at least 2 carbon atoms in the linking chain, 
and consisting of a combination of at least two of alkylene, having 1 to 8 
carbon atoms, arylene, having about 6 to 12 carbon atoms, hetero atoms, 
and heteroatom-containing groups, 
m is 0 or 1, n is 1 or 2, and l is 0 or 1, with the proviso that when n is 
2, one of the alkylene and arylene is necessarily trivalent. 
More specifically, in the structure noted above, R is hydrogen, alkyl of 1 
to 3 carbon atoms (such as methyl, ethyl, isopropyl and n-propyl), or halo 
(such as chloro or bromo). Preferably, R is hydrogen, methyl or chloro. 
More preferably, R is hydrogen or methyl. 
Also, L is an organic linking group having at least 2 carbon atoms in the 
linking chain and is a combination of at least two of (1) alkylene groups 
having 1 to 8 carbon atoms, such as methylene, ethylene, trimethylene, 
propylene, pentamethylene, or 2,2-dimethyl-1,3-propylene, (2) arylene 
groups having 6 to 12 carbon atoms, such as phenylene, tolyene, xylylene, 
naphthylene, and (3) divalent hetero atoms, such as oxygen (oxy), and 
sulfur (thio) atoms, or (4) heteroatom-containing groups, such as 
carbonyl, sulfonyl, imino, (--NR.sup.1 where R is hydrogen or lower alkyl 
of 1 or 6 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl and 
hexyl). 
The alkylene groups can have from 1 to 8 carbon atoms, and can be branched, 
linear or cyclical, substituted or unsubstituted with one or more alkyl 
groups (preferably of from 1 to 8 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-methoxyphenyl and chlorophenyl). Such groups are 
not difficult to design or synthesize for one skilled in synthetic 
chemistry. The arylene groups can have from about 6 to 12 nuclear carbon 
atoms and can have substituents as defined for the alkylene groups. 
Preferably, L comprises alkyleneoxycarbonylalkylene, 
alkylenethioalkyleneoxycarbonylalkylene, alkyleneiminocarbonylalkylene, 
iminoalkyleneoxycarbonylalkylene, alkylenethioalkylene, 
alkylenethioalkyleneiminocarbonylalkyleneoxyalkylene, 
alkylenethioalkylidyne, 
alkylenethioalkyleneiminocarbonylalkylenethioalkylene, 
alkylenethioalkyleneiminocarbonylalkylene, alkylenethioarylene, 
alkylenethioalkyleneoxyalkylenethioalkyleneoxycarbonylalkylene, 
alkyleneoxyarylenealkylenethioalkylene, 
alkylenethioalkyleneoxyalkylenethoalkyleneoxycarbonylalkylene, 
alkyleneoxyrylenealkylenethioarylenealkylenethioalkylene, 
alkylenethioalkyleneoxyalkylenethioalkyleneoxycarbonylarylene, 
carbonyloxyalkyleneoxycarbonylalkylene, 
carbonyloxyalkyleneureylenealkylene, 
carbonyloxyalkyleneiminocarbonylalkylene, or 
carbonyloxyalkyleneoxycarbonylalkylene. 
More preferably, l is 1 and L is alkylenethioalkylene, 
alkylenethiophenylene, or alkylenethiophenylidyne. 
Representative L groups include, but are not limited to: 
methyleneoxycarbonyltrimethylene, 
methylenethioethyleneoxycarbonyltrimethylene, 
methyleneiminocarbonyltrimethylene, 
methylene-N-methyliminoethyleneoxycarbonyltrimethylene, 
methylenethioethylene, 
methylenethioethyleneiminocarbonylmethyleneoxymethylene, 
methylenethio-1,1,2-ethylidynemethylenethioethyleneiminocarbonylmethylenet 
hiomethylene, methylenethioethyleneiminocarbonyltrimethylene, 
methylenethio-1-carboxyethylene, methylenethiophenylene, 
methylenethioethyleneoxyethylenethiomethyleneoxycarbonylethylene, 
methyleneoxyphenylenemethylenethioethylene, 
methylenethioethyleneoxyethylenethioethyleneoxycarbonylethylene, 
methyleneoxyphenylenemethylenethiophenylenemethylenethiotrimethylene and 
methylenethioethyleneoxyethylenethioethyleneoxycarbonylphenylene. 
Also, m is 0 or 1, n is 1 or 2, and l is 0 or 1, with the proviso that when 
n is 2, one of said alkylene and arylene is necessarily trivalent. 
Most preferably, l and m are 0. 
Preferably, monomer (b) is styrene or a styrene derivative, or an acrylic 
or methacrylic acid ester. More preferably, it is 
N-acryloyloxysuccinimide, 4-(2-succinimidoxycarbonylethylthiomethyl)styren 
e, 4-[1,2-bis(succinimidoxycarbonyl)ethylthiomethyl]styrene, or 
4-(2-succinimidoxycarbonylphenylthiomethyl)styrene. 
While the monomers (b) described above can be polymerized to form 
homopolymers, preferably they are used to prepare copolymers with one or 
more additional ethylenically unsaturated polymerizable monomers. For 
instance, 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. 
Monomers from which (a) can be derived include, but are not limited to, 
vinyl aromatics (for example, styrene and styrene derivatives such as 
4-vinyltoluene, .alpha.-methylstyrene, 2,5-dimethylstyrene, 
4-t-butylstyrene and 2-chlorostyrene), acrylic and methacrylic acid esters 
and amides (for example, methyl acrylate, methyl methacrylate, n-butyl 
acrylate, 2-ethylhexyl methacrylate, benzyl acrylate and 
N-phenylacrylamide), butadiene, acrylonitrile, vinyl acetate, vinylbenzyl 
acetate, vinyl bromide, vinylidene chloride and others readily apparent to 
one skilled in the art. Preferred (a) monomers are the vinyl aromatics 
with styrene being most preferred. 
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-acrylamido-2-methylpropane sulfonic acid, 
3-methacryloyloxypropane-1-sulfonic acid, p-styrenesulfonic 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, pentaethylene glycol 
monomethacrylate, 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 could be 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 present herein. 
The copolymers of this invention are water insoluble and are readily formed 
as particles. The preferred monomers useful in the method of making the 
copolymers of this invention polymerize readily with styrene. Styrene has 
a low solubility in water. 
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 Polymer Science, 2nd Ed. (1968), Wiley and 
Sons, New York, and Stevens, Polymer Chemistry, An Introduction, Addison 
Wesley Publishing Co., London, 1975, and certain preferred conditions are 
discussed in copending U.S. Pat. No. 07/646,132 of Sutton et al (noted 
above). 
During polymerization, poragens (pore-producing substance) or inert 
diluents are not used. If used, they would normally result in the 
formation of pores. If porosity was required and pore integrity is to be 
maintained, the particles are typically crosslinked so that they do not 
dissolve in the polymerization solvent or the inert diluent or poragen. 
Therefore, the particles of this invention are non-porous and do not 
require a crosslinked structure to be useful. 
The copolymers described herein are critically 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 from about 
0.01 to about 20 .mu.m, preferably from about 0.01 to about 10 .mu.m, and 
more preferably from about 0.05 to about 5 .mu.m. 
The reagents prepared by the method of this invention have one or more 
biologically active substances having been covalently attached to the 
polymeric particles through amide groups by displacement of the active 
succinimid ester 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 on which 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 occurring in biological fluids. Such materials must be capable 
of attachment to the particles by displacement of the active succinimidoxy 
groups. Thus, generally, this means that the biologically active substance 
has an available amino or sulfhydryl group for reaction. 
Depending upon the intended use of the reagent, the biologically active 
substances include, but are not limited to, amino acids, peptides, 
polypeptides, proteins (including antibodies, C-active protein and avidin 
and its derivatives), lipoproteins, glycoproteins, hormones, drugs (for 
example digoxin, phenytoin, phenobarbital, thyroxine, triiodothyronine, 
gentamicin, carbamazepine and theophylline), steroids, vitamins, 
polysaccharides, glycolipids, alkaloids, microorganisms, viruses, 
protozoa, fungi, parasites, rickettsia, molds, blood components, tissue 
and organ components, pharmaceuticals, haptens, lectins, toxins, nucleic 
acids (including oligonucleotides, either single- and double-stranded), 
antigenic materials (including proteins and carbohydrates), avidin or 
derivatives thereof, 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 prepared by the method 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 
detail below). Examples of ligand-receptor complexes (that is, reaction 
products of 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 oligonucleotides complementary thereto at 
different sequences. 
Most useful biologically active substances are what are known in the art as 
immunoactive 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 and auto-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 
active site for reaction with the active succinimidoxycarbonyl groups on 
the particles. 
Particularly useful biologically active substances include antibodies 
directed to Streptococcus A, HIV-I, a microorganism associated with 
periodontal disease, carbamazepine, thyroxine, human chorionic 
gonadotropin, phenobarbital, phenytoin, digoxin or a C-active protein. 
In certain embodiments, the immunological species is an enzyme which has a 
active group for attachment. Representative enzymes include, but are not 
limited to, horseradish peroxidase, glucose oxidase, urease, 
.beta.-galactosidase, aspartate aminotransaminase, alanine 
aminotransaminase, lactate dehydrogenase, creatine phosphokinase, 
.UPSILON.-glutamyl transferase, alkaline phosphatase, acid phosphatase and 
prostatic acid phosphatase. 
In 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 active 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 U.S. Pat. No. 4,914,210 (issued 
Apr. 3, 1990 to Levenson et al) and WO-A-89/2932 (published Apr. 6, 1989), 
both directed to modification of oligonucleotides, U.S. Pat. No. 4,719,182 
(issued Jan. 12, 1988 to Burdick et al), Erlanger et al, J. Biol. Chem., 
234, 1090 (1959), Wiston et al, Biochim. Biophys. Acta, 612, pp. 40-49 
(1980) and Borzini et al, J. Immunol. Methods, 44, pp. 323-332 (1981). 
The procedure for attaching a biologically active substance to polymeric 
particles to prepare the reagents of this invention is generally as 
follows. 
An aqueous suspension of the polymer particles is directly treated with a 
buffered solution or suspension of the biologically active material to be 
having been covalently bound to the polymer particles in a weight ratio of 
biologically active substance to polymer of about 0.1:1000 to about 1:1, 
preferably from about 1.0:1000 to about 100:1000, and a pH ranging from 
about 7.0 to about 10.0, preferably from about 8.5 to about 9.5. The 
mixture is mixed for a time sufficient for reaction completion, that is, 
as much as several hours, although from 2 to 28 hours is generally 
suitable. If necessary, the reactions are quenched with bovine serum 
albumin (BSA), then the particulates isolated (preferably by 
centrifugation), and resuspended in buffered saline. The isolation and 
resuspension steps are repeated one or more times and the final dispersion 
is prepared at about 0.1 to 40, and preferably 1 to 10 weight percent 
solids and stabilized (preferably with about 0.02% merthiolate). One 
skilled in the art can vary this procedure considerably, particularly the 
times, temperature, buffers, stabilizers, or other reagents, isolation or 
quenching techniques with equivalent results. 
In the reaction mixture, the % solids of particles is generally from about 
0.01 to about 40%, and preferably from about 1.0 to about 10%, in 
preparing the reagent. The amount of biologically active substance is 
generally designated by a weight ratio of substance to copolymer of from 
about 0.1:1000 to about 1:1, and preferably from about 1.0:1000 to about 
100:1000. However, it should be understood that not all of the substance 
may become having been covalently bound to the particles. In fact, a minor 
amount may be adsorbed, and some may not be bound at all. One skilled in 
the art could readily perform tests to determine the amount of substance 
bound to the particles. Hence, usually an excess of substance is mixed 
with the particles than actually becomes covalently bound. 
Mixing of the biologically active substance and particles is carried out at 
a temperature of from about 5 to about 50.degree. C. preferably 18.degree. 
C. to 40.degree. C., for from about 2 to about 28 hours, preferably 4 to 
24 hours. The length of time will vary with the temperature, biologically 
active substance and the desired coverage. Any suitable buffer can be 
used, but 4-(2-hydroxyethyl)-1-piperazinopropanesulfonic acid is 
preferred. 
The details of representative procedures for making various reagents are 
shown in the Examples below. 
The nucleic acid reagents are advantageously prepared similar to the other 
reagents described above, but more particularly, the polymeric particles 
having an average particle size of from about 0.01 to about 10 .mu.m are 
present in the suspension in an amount of at least about 1% solids, and 
preferably from about 5% to about 25% solids. The advantage of this 
feature is that is produces a reagent that gives a much higher signal in 
an assay for cytomegaloviral DNA. 
Generally, the method of preparing the nucleic acid reagents comprises: 
(A) contacting (1) an aqueous suspension of succinimid-containing polymeric 
particles having an average particle size of 0.05 to about 10 .mu.m, the 
particles being present therein at at least about 1% solids with an 
oligonucleotide having an active amine or sulfhydryl group which reacts 
with the succinimidoxycarbonyl active groups to form a covalent linkage 
between the particles and the oligonucleotide. 
Where the oligonucleotide does not have the requisite active amine or 
sulfhydryl groups, they can be added using known procedures and reactants 
as described, for example, in U.S. Pat. No. 4,914,210 (issued Apr. 3, 1990 
to Levenson et al.). 
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 specifically active 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 fluids (such as 
biological fluids), 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 fluids of humans including whole blood, sera, plasma, lymph, 
bile, urine, spinal fluid, sputum, lacrimal fluid, perspiration, swab 
specimens, tissue cultures, stool secretions, cellular fluids, vaginal 
secretions and semen. It is also 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 antibodies thereto (such as Streptococcus A antigen, 
Chlamydial or Gonococcal antigen or antibody), viruses or components 
thereof (such as hepatitis, cytomegalovirus or herpes antigen) or 
antibodies thereto, cancer-producing agents, or C-active 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 of HIV-I DNA, 
.beta.-globin DNA or cytomegaloviral DNA is of particular interest in the 
practice of this invention. 
In general, the method for the determination of a specific binding ligand 
comprises: 
A. contacting a specimen suspected of containing a specific binding ligand 
with a reagent comprising: 
(I) a particle as described above, and 
(II) a receptor for said ligand having been covalently attached to said 
particle through an amide group by displacement of the succinimidoxy 
portion of said succinimidoxycarbonyl group, 
to form a water-insoluble specific binding complex of said ligand with said 
receptor, and 
B. detecting the presence of said complex as an indication of the presence 
or absence of said ligand in said specimen. 
The ligand is preferably an antigenic material, hapten or drug. The 
receptor is preferably an antibody for said antigenic material, hapten or 
drug. 
In another embodiment, the reagent can be used in competitive binding 
assays for determination of a water-soluble specific binding ligand. In 
general, such an assay comprises: 
A. contacting a specimen suspected of containing a water-soluble specific 
binding ligand with a reagent comprising: 
(I) a particle as described above, and 
(II) molecules of the ligand having been covalently attached to the 
particle through an amide group by displacement of the succinimidoxy 
portion of the succinimidoxycarbonyl group. 
B. prior to, simultaneously with or subsequent to said contact in step A, 
contacting said specimen with a receptor for said ligand to form a 
water-soluble specific binding complex of said receptor with said 
water-soluble ligand and a water-insoluble specific binding complex of 
said receptor with said water-insoluble ligand, 
C. separating said water-insoluble complex from water-soluble materials, 
and 
D. detecting the presence of said water-insoluble complex as an indication 
of the presence or absence of said ligand in said specimen. 
Such competitive binding assays can be carried 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 interest. Either 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 so that 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 of this invention can also be carried out using dry analytical 
elements. The simplest element can be composed of an adsorbent, fluid 
permeable substrate, for example, a thin sheet of a self-supporting 
adsorbent or bibulous 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 U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to 
Przybylowicz et al), U.S. Pat. No. 4,258,001 (issued Mar. 24, 1981 to 
Pierce et al), U.S. Pat. No. 4,292,272 (issued Sep. 29, 1981 to Kitajima 
et al) and U.S. Pat. No. 4,430,436 (issued Feb. 7, 1984 to Koyama et al). 
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 can 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 
for 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 U.S. Pat. No. 4,042,335 
(issued Aug. 16, 1977 to Clement), U.S. Pat. No. 4,132,528 (issued Jan. 2, 
1979 to Eikenberry et al), U.S. Pat. No. 4,144,306 (issued Mar. 13, 1979 
to Figueras), U.S. Pat. No. 4,670,381 (issued Jun. 2, 1987 to Frickey et 
al) and EP-A-0 253 581 (published Jan. 2, 1988). 
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 element at the time of the assay along with the test 
specimen. Preferably, however, the ligand analog and reagent of this 
invention (containing the appropriate receptor) are located within the 
element in different zones so they will not complex prematurely. 
This invention also provides an analytical element comprises 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) the particle as described above, and 
(II) a biologically active substance having been covalently attached to the 
particle through an amide group by displacement of the succinimidoxy 
portion of a succinimidoxycarbonyl group. 
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 in response to an enzyme, 
a water-soluble layer containing an analog of a ligand labeled with said 
enzyme, and 
a porous spreading layer containing a biologically active reagent 
comprising: (I) a water-insoluble, nonporous particle of a noncrosslinked 
copolymer as previously described herein, and (II) a receptor for the 
ligand having been covalently attached to the particle through an amide 
group by displacement of the succinimidoxy portion of a 
succinimidoxycarbonyl group. 
Preferably, the ligand analog is labeled with an enzyme, such as one 
described below, the ligand is an antigenic material, hormone, hapten or 
drug, and the receptor is the corresponding antibody or immunoreactant. 
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 variety 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 contact can be accomplished in any suitable manner, for example, by 
dipping or immersing the element into the sample or, preferably, by 
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 U.S. Pat. No. 4,517,288 (issued May 14, 1985 to Giegel et al). 
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 clumping of the 
particles. The resulting 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 U.S. Pat. No. 4,419,453 (issued Dec. 6, 1983 to Dorman et al), 
U.S. Pat. No. 4,808,524 (issued Feb. 28, 1989 to Snyder et al), U.S. Pat. 
No. 4,828,978 (issued May 9, 1989 to Warren III et al) and U.S. Pat. No. 
4,847,199 (issued Jul. 11, 1989 to Snyder et al). 
Agglutination assays are preferably 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 
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 U.S. Pat. No. 4,808,524 (noted above) and in EP-A-0 280 556 (published 
Aug. 31, 1988). In core-shell polymer particles, the shell copolymer has a 
composition like that described herein (that is, having the necessary 
active succinimidoxycarbonyl groups), but the core polymer can be 
different and need not have active groups. 
Also, a method for the determination of an immunological species is 
provided comprising: 
A. contacting a specimen suspected of containing an immunological species 
with a reagent of this invention having a receptor for the species which 
is having been covalently attached to the particle through an amido group 
by displacement of the succinimidoxy portion of the succinimidoxycarbonyl 
group, to form a water-insoluble immunological complex of the species with 
the receptor, removing water-soluble materials from the complex preferably 
by filtration, and detecting the presence of the complex as an indicator 
of the presence or amount of the immunological species in the specimen. It 
is preferred that the reagent be immobilized on a microporous filtration 
membrane. 
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. 
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 different), one 
of which is insolubilized or capable of being insolubilized (such as 
through an avidin-biotin 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 complex is insoluble, detectable and separatable from 
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 GB-A-2,074,727 (published Nov. 
4, 1981) and U.S. Pat. No. 4,486,530 (issued Dec. 4, 1984 to David et al), 
and references noted therein. 
Preferably, in the sandwich assays, either prior to, simultaneously with or 
subsequently to the 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 active 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 the sandwich assay, the reagent of this invention can be directly 
reacted with the ligand of interest, for example, where the ligand is an 
antigen, and the reagent comprises antibodies thereto. Alternatively, the 
reagent is complexed with the ligand indirectly, that is, through an 
intermediate linking moiety. One example of this is shown in U.S. Pat. No. 
4,870,007 (issued Sep. 26, 1989 to Smith-Lewis) 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 U.S. Pat. No. 
4,358,535 (issued Nov. 9, 1982 to Falkow et al) and U.S. Pat. No. 
4,486,539 (issued Dec. 4, 1984 to Ranki et al). These reagents can also be 
used as capture probes after what is known in the art as polymerase chain 
reaction amplification, for example, as described in more detail in U.S. 
Pat. No. 4,683,195 (issued Jul. 28, 1987 to Mullis et al), U.S. Pat. No. 
4,683,202 (issued Jul. 28, 1987 to Mullis) and EP-A-0 370,694 (published 
May 30, 1990). An amplified nucleic acid is immobilized by hydridization 
with the reagent of this invention. 
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 comprising an 
oligonucleotide having been covalently attached to the particle through 
the active succinimidoxycarbonyl group, 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) with suitable 
reagents (for example, DNA polymerase, dNTPs, primers) prior to capture 
with the reagent of this invention. HIV-I DNA, cytomegaloviral DNA and 
.beta.-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 amplified 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 commercially available from Pall 
Corp.). Representative test devices are shown in U.S. Pat. No. 3,825,410 
(issued Jul. 23, 1974 to Bagshawe), U.S. Pat. No. 3,970,429 (issued Jul. 
20, 1976 to Updike) and U.S. Pat. No. 4,446,232 (issued May 1, 1984 to 
Liotta). Particularly useful test devices are shown in U.S. Ser. No. 
98,248 (filed Sep. 18, 1987 by Hinckley et al), and are commercially 
available as Surecell.TM. 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 of biological materials. 
Thus, the reagent 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 
chromatography. 
Affinity chromatography can also be used to concentrate dilute solutions of 
proteins in order to remove denatured forms thereof from refined proteins, 
and in the separation and resolution of protein and peptide components 
which have originated in specific chemical modifications. 
Another use of this method is to purify nucleic acids, such as those 
resulting from polymerase chain reaction amplification, as described, for 
example in EP-A-O 388,171 (published Sep. 19, 1990). 
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 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. 
The analytical purification method of this invention comprises (A) passing 
a specimen containing a mixture of biologically active substances over an 
affinity chromatography reagent comprising the particles described above, 
and a specific binding substance having been covalently attached to said 
particle through an amide group by displacement of the succinimidoxy 
portion of the succinimidoxycarbonyl group, with the specific binding 
substance being specific to one or more predetermined biologically active 
substances in the specimen mixture of biologically active substances, and 
collecting the one or more predetermined biologically active substances on 
the reagent. 
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 to the reagent and replace the captured 
substance. 
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 
Immobilization of anti-phenobarbital antibody Phe 1.9 on polymer beads of 
the invention and retention of antibody activity 
The following copolymer beads were evaluated: 
______________________________________ 
SAMPLE COMPOSITION 
______________________________________ 
1 Poly(styrene-co-N-acryloyloxysuccinimid) 
(Molar ratio 96.83/3.17; Weight ratio 85/5) 
2 A core/shell polymer having a core of 
poly(styrene-co-ethylene dimethacrylate) 
(molar ratio 99/1) and a shell of 
poly(styrene-co-m- and 
-p-(60/40)-(2-chloroethylsulfonylmethyl)styrene- 
--co-ethylene dimethacrylate) (molar ratio 
94.5/4.5/1) 
3 Poly(styrene- --co- .sub.-- m- and -p-(60/40)-2-(2- 
chloroethylsulfonylmethyl)styrene- --co- 
methacrylic acid) (molar ratio 
89.14/4.16/6.34) 
______________________________________ 
The beads were all treated under the same condition. A reaction dispersion 
was prepared comprising 0.3 mg of anti-phenobarbital antibody 1.9 and 30 
mg dry weight of beads in 0.1M EPPS buffer 
[N-(2-hydroxyethyl)piperazine-N'-(3-propane-sulfonic acid)], pH 8.5, 1.5 
ml final volume and placed in tubes. The capped tubes containing the 
reaction mixtures were rotated end-over-end at room temperature for 4 
hours. The reactions were quenched by the addition of 0.3 ml of bovine 
serum albumin (BSA) solution at 100 mg/l. The tubes were rotated 
end-over-end for an additional 16 hours at room temperature. The reaction 
mixtures were centrifuged, the supernatants removed, and the beads 
resuspended in 1 ml of PBS (phosphate buffered saline, pH 7.4). This step 
was repeated 3 more times. The final resuspension was in 1.8 ml PBS, and 
merthiolate was added to 0.02% final concentration. 
Identical reactions were performed using .sup.125 I-labeled BGG (bovine 
gamma globulin). These reactions were performed to assess the total mass 
of antibody adsorbed to the surface. The analysis was performed as 
follows: a quantity of 0.18 ml of the reaction mixture following addition 
of the BSA quench was taken and monitored for radioactivity. As in the 
case of the Phe 1.9 beads, the beads were centrifuged; a 0.18 ml sample of 
the supernatant was taken and monitored for radioactivity. The remainder 
of the supernatant was removed and the beads were resuspended in 1 ml of 
PBS. The beads were centrifuged once more. The beads were resuspended in 1 
ml of 1% sodium dodecyl sulfate and monitored for radioactivity. The ratio 
of radioactivity of the washed beads to the initial radioactivity of the 
reaction mixture was used to calculate the amount of .sup.125 I-BGG, and 
by analogy the amount of Phe 1.9 antibody, adsorbed on the bead surfaces. 
The relative amounts of active antibody in the preparations were determined 
in an assay in which serial dilutions of the antibody bead dispersions 
were mixed with a fixed concentration of one of two antigens: 
phenobarbital-enzyme label or .sup.3 H-phenobarbital. The reaction between 
the diluted antibody bead dispersions and the enzyme labels were incubated 
for one hour at room temperature with constant agitation in PBS containing 
0.1% BSA. Following incubation, the beads were centrifuged and a sample of 
the supernatant was removed to determine the concentration of antigen 
remaining in solution. The antibody binding sites required to bind 50% of 
the antigens were calculated. The results are summarized below: 
______________________________________ 
Nanomolar theoretical binding sites 
required to remove 50% of the antigen 
enzyme-label 
.sup.3 H-Phenobarbital 
Sample at 5e-10M total 
at 5e-9M total 
______________________________________ 
1 0.60 11 
2 1.4 25 
3 1.0 25 
______________________________________ 
These results show that Phe 1.9 immobilized on the beads of Sample 1 can 
bind more phenobarbital-enzyme conjugate or phenobarbital drug at lower 
concentrations of immobilized Phe 1.9 indicating that Phe 1.9 immobilized 
on said beads retains more activity than when immobilized on the other 
beads. 
EXAMPLE 2 
Immobilization of anti-phenytoin antibody DPH 1.2 on copolymer beads of 
this invention and retention of activity 
The following copolymer beads were evaluated. 
______________________________________ 
SAMPLE COMPOSITION 
______________________________________ 
1 Same polymer as in Example A, sample 1, 
except having a molar ratio of 87.32/12.68 
rather than 96.83/3.17 
2 Same polymer as Example A, Sample 2 
3 Poly(styrene-co-m- and p-(60/40)-(2- 
chloroethylsulfonylmethyl)styrene-co- 
ethylene dimethacrylate) (molar ratio 
94.5/4.5/1) 
4 Poly(styrene-co-acrylic acid) (molar ratio 
97.5/2.5) 
5 Poly(styrene-co-m- and p-(60/40)-(4- 
carboxybutyryloxymethyl)styrene) 
(molar ratio 97.84/2.16) 
6 Poly(styrene-co-4-carboxybutyryloxyethyl 
methacrylate) (molar ratio 97.84/2.16) 
7 Poly(styrene-co-4-carboxybutyrylpoly- 
(oxyethylene) methacrylate (molar ratio 
98.71/2.19) 
______________________________________ 
The activator for the reaction of the lysine residues of the antibody with 
the carboxylic acid groups of copolymers 4-7 is 
1-(1-pyrrolidinylcarbonyl)pyridinium chloride (DS-1). 
The beads were all treated under the same conditions. Reaction dispersions 
were prepared comprising 0.3 mg of antibody (anti-phenytoin antibody DPH 
1.2) and 30 mg dry beads and a final volume of 1.5 ml. For beads 1-3, 0.1M 
EPPS buffer, pH 8.5 was used. For beads 4-7, 0.1M MES 
(2-(4-morpholino)ethanesulfonic acid) was used and 0.5 mmol of DS-1 
activator/g beads (0.015 mmol) was present. The antibody bearing beads 
were prepared as follows: 
Aliquots of the polymer particle dispersions containing 30 mg beads dry 
weight were placed in 2 ml microfuge tubes. The appropriate buffers were 
added to bring the volume in each tube to 1.5 ml. The beads were 
centrifuged 10 min. at 13,000 rpm and the supernatant discarded. Beads 1-3 
were resuspended with 1.393 ml of 0.1M EPPS buffer. Beads 4-7 were 
resuspended with 1.093 ml of 0.1M MES buffer. A solution of DS-1 (0.3 ml 
of a solution at 0.05M in 0.1M MES) was added to tubes 4-7; the tubes were 
capped and rotated end-over-end for 10 minutes. An aliquot (0.107 ml at 
2.82 mg/ml) anti-phenytoin antibody DPH 1.2 was added to each tube, the 
tubes were capped and rotated end-over-end for 4 hours. 
The reactions were quenched by the addition of 0.3 ml of bovine serum 
albumin solution at 100 mg/ml. The tubes were rotated end-over-end for an 
additional 16 hours at room temperature. The reaction mixtures were 
centrifuged, the supernatants removed and saved for analysis, and the 
beads resuspended in 1 ml of PBS. This step was repeated 3 more times. The 
final resuspension was in 1.8 ml PBS and merthiolate was added to 0.02% 
final concentration. 
The supernatants from the reaction mixture were analyzed for total antibody 
concentration by ELISA. The amount of antibody bound to the surface was 
calculated from this result. 
The relative amounts of active antibody in the preparations were determined 
in an assay in which serial dilutions of the antibody bead dispersion were 
mixed with a fixed concentration of one of three different antigens: 
hapten-enzyme conjugates prepared with two different haptens 
(5,5-diphenylhydantoin-3-.omega.-valeric acid (DPH-val-) and 
5-ethyl-5-phenylhydantoin-3-.omega.-valeric acid (EPH-val) and .sup.3 
H-phenytoin. The reaction between the diluted antibody head dispersions 
and the various antigens were incubated for one hour at room temperature 
with constant agitation in PBS containing 1.0% BSA. The amount of 
enzyme-label or .sup.3 H phenytoin remaining in solution after 
centrifugation was determined and the theoretical number of binding sites 
required to bind 50% of the antigens were calculated. The results are show 
below: 
______________________________________ 
Nanomolar theoretical binding sites required 
to bind 50% of the antigen at 5e-10M total 
Sample 
DPH-val-enzyme 
EPH-val-enzyme 
.sup.3 H-phenytoin 
______________________________________ 
1 2.5 2.5 1.2 
2 2.5 4.0 1.2 
3 2.0 5.0 1.1 
4 10 120 12 
5 15 120 6.0 
6 10 80 6.0 
7 25 &gt;250 15 
______________________________________ 
These results show DPH 1.2 immobilized on beads of Sample 1 binds 
EPH-val-enzyme at lower concentrations of immobilized binding sites than 
DPH 1.2 immobilized on any of the other beads. This indicates that DPH 1.2 
retains more EPH-val-enzyme binding sites when immobilized on beads of the 
invention than when immobilized on the other beads. DPH 1.2 retains more 
activity toward the antigen when immobilized on beads of Sample 1 than 
when immobilized on any of the carboxylic acid group bearing beads. DPH 
1.2 activity toward DPH-val-enzyme and .sup.3 H-phenytoin when immobilized 
on Sample 1 beads is similar to the activity of DPH 1.2 immobilized on the 
type beads which have chloroethylsulfonyl groups. 
EXAMPLE 3 
Reaction of the Particles of Example 1, Sample 1 with .sup.3 H BGG 
One portion of Example 1, Sample 1 (93 .mu.L, 16.2%) solids containing 15 
mg of polymer (dry weight) was combined with 27 .mu.L at 10 mg/ml (0.273 
mg) of labeled (tritiated) bovine gamma globulin (.sup.3 H BGG) and 
brought to a final volume of 1.5 ml with 0.1M EPPS, pH 8.5, in a 
microcentrifuge tube. [EPPS=4-(2-hydroxyethyl)-1-piperazinopropanesulfonic 
acid.] The reaction was continued for 24 hours at room temperature by 
end-over-end rotation of the tube at 30-35 rpm while attached to a 
rotating plate mounted at a 45.degree. angle. A second portion was treated 
as described above except that the reaction was conducted at 37.degree. C. 
At the end of the described reaction times, each reaction was quenched by 
addition of excess BSA, bovine serum albumin (250 .mu.L of BSA at 100 
mg/ml) and continuing rotation for an additional 4 hours. 
The total amount of .sup.3 H BGG bound was determined by measuring: a) the 
cpm (counts per minute) of a 250 .mu.L aliquot of the reaction mixture; b) 
the cpm of a 250 ml aliquot of the supernatant following centrifugation at 
14,000 rpm, 10 min, of a 1 ml sample of the reaction mixture; and c) the 
cpm of the material bound to the latex following repeated washes of the 
pellet obtained in (b). The fraction of BGG which is having been 
covalently bound to the latex was determined following incubation of a 500 
.mu.L reacted latex in the presence of 400 .mu.L of 0.1M EPPS buffer and 
100 .mu.L of 10% sodium dodecyl sulfate (SDS) at 37.degree. C. for 20 
hours with end-over-end rotation. The same procedure described above for 
determining the total amount of .sup.3 H BGG bound is used to determine 
the amount having been covalently bound. The results are reported in 
Tables I and II. 
TABLE I 
______________________________________ 
BGG Bound to Polymer 
SAM- REACTION .sup.3 H BGG 
% mg BGG/g 
PLE TEMP. OFFERED BOUND POLYMER 
______________________________________ 
1 Room Temp 0.5 mg 82 2.7 
2 37.degree. C. 
0.5 mg 85 2.8 
______________________________________ 
TABLE II 
______________________________________ 
.sup.3 H BGG Covalently Bound (After SDS Treatment) 
RATIO 
SAM- REACTION % mg BGG/g COVALENT/ 
PLE TEMP BOUND POLYMER TOTAL 
______________________________________ 
1 Room Temp 57 1.9 .69 
2 37.degree. C. 
60 2.0 .71 
______________________________________ 
The results of these reactions show that roughly equivalent amounts of 
.sup.3 H BGG are having been covalently attached at either temperature. 
The invention has been described in detail with particular reference to 
certain 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.