High refractive index haloalkyl-functional shell-core polymers and their use in light scattering immunoassays

Novel particle reagent for light scattering immunoassays are provided. The particle reagents are high refractive index shell-core polymers, having at least a partial surface coverage by a monomolecular layer of anionic surfactant, covalently bonded to compounds of biological interest. The novel particle reagents are particularly suited to protein immobilization by covalent bonding to the shell and are especially useful for light scattering immunoassays.

TECHNICAL FIELD 
This invention relates to novel particle reagents based on shell-core 
particles in which the high refractive index of the core results in high 
sensitivity light scattering measurements and the shell contains a polymer 
of chloromethylstyrene. 
BACKGROUND ART 
Agglutination reactions are known to produce aggregates that can be 
visually or instrumentally detected for qualitative or quantitative assay 
of biological materials. Agglutination reactions for antigens usually 
involve antibodies or other binding agents having at least two combining 
sites specific for multiple, complementary sites on their corresponding 
antigens or binding partners. These antigens can be found associated with 
bacterial and mammalian cell surfaces, viral capsids and envelopes, and as 
soluble and insoluble materials of biological interest such as proteins, 
carbohydrates, and nucleic acids. Alternatively, agglutination assays for 
antibodies having a minimum of two antigen-reactive sites are accomplished 
by adding the multivalent antigen to a solution containing the antibodies. 
Also useful are agglutination inhibition assays in which a known quantity 
of labelled, multiepitopic antigen or multivalent antibody competes with 
an unknown quantity of antigen or antibody, respectively, for combining 
sites on the binding partner, thereby reducing the extent of 
agglutination. 
The extent of agglutination reactions is known to depend upon the relative 
concentrations of binding partners. Optimal concentration ranges for each 
can be empirically determined to provide conditions under which extensive 
cross-linking of reactants occurs. This cross-linking of individual 
binding partners provides efficient light scattering aggregates, which can 
be visually or spectrophotometrically detected. A molar excess of either 
binding partner relative to the other diminishes or eliminates the 
cross-linking agglutination reaction in a phenomenon known as the prozone 
effect, which reduces assay sensitivity. Therefore, maximum assay 
sensitivity is assured by adjustment of binding partner concentrations 
into optimal ranges as determined by maximal changes in the light 
scattering properties of an agglutination or agglutination inhibition 
reaction. 
Sensitivity enhancement has been achieved in agglutination-based 
immunoassays by attaching immunoreagents to particulates whose improved 
light scattering efficiency provides additional changes in light 
scattering signal for each agglutination event. Immunoreagents have been 
adsorbed onto particulate materials such as latex shperes, or covalently 
bonded to specific functional groups on particle surfaces for improved 
stability. U.S. Pat. No. 4,064,080, issued Dec. 20, 1977, discloses 
styrene polymers with terminal aminophenyl groups having proteins attached 
to them. U.S. Pat. No. 4,181,636, issued Jan. 1, 1980, discloses 
carboxylated latex polymers coupled to immunologically active materials 
through a water soluble activating agent and their use as diagnostic 
reagents in agglutination tests. 
Improved latex reagents are disclosed in U.S. Pat. No. 4,401,765, issued 
Aug. 30, 1983, comprising shell-core latex polymer particles of 0.03-0.1 
.mu.m diameters having a high refractive index polymer core and a polymer 
shell containing reactive groups such as epoxy, carboxyl, amino, hydroxyl, 
or formyl groups for covalent coupling of proteins. These improved latexes 
provide a high refractive index core which maximizes light scattering 
efficiency while also providing selected functional groups for hapten and 
protein immobilization onto reactive shells. 
Core-shell latex particles of 0.05-1.0 .mu.m diameters having active 
halogen monomers copolymerized with other ethylenically unsaturated 
monomers in the particle shell are disclosed in U.S. Pat. No. 4,017,442, 
issued Apr. 12, 1977. These particles are characterized by having cationic 
or nonionic surfaces generated by use of surfactants during or after 
particle polymerizations. Microparticles comprising monodisperse latex 
beads of 0.5 .mu.m mean diameter and containing copolymerized 
chloromethylstyrene are commercially available from Polysciences, Inc., 
Warrington, PA., 18976, for use with covalently bound antigens and 
antibodies in agglutination tests. These particles are made from styrene 
copolymerized with chloromethylstyrene and cross-linked with 
divinylbenzene. U.S. Pat. No. 4,056,501, issued Nov. 1, 1977, discloses 
further treatment of the particle reagents prepared according to U.S. Pat. 
No. 4,017,442, previously cited, with nucleophilic groups such as 
dialkylsulfides or quaternary amines to react with the halogenated latexes 
to form stable, dispersed particle suspensions in aqueous media. These 
suspensions were shown to be useful for coatings and organic pigments. 
Many of the functional groups found on particle reagents of the prior art 
are often less than optimal for protein immobilization. For example, some 
polymeric latex particles having functional groups such as carboxyl and 
amino groups, require activation prior to protein coupling. The outcome of 
such a process is variable with respect to the degree of activation and 
the stability of proteins once they are attached. In other cases such as 
with epoxy groups, activation is not required, but the active functional 
groups on the particles are hydrolytically unstable. This results in 
variably reactive particles that can provide a low concentration of 
covalently attached proteins. Finally, with functional groups which are 
either themselves reactive (autoreactive) or require an activation 
process, the protein coupling conditions may be too stringent requiring 
the use of elevated temperatures over long periods in the presence of 
surfactants to effect covalent protein attachment to particles. Such 
conditions can result in protein denaturations, which can be tolerated 
only if antigenic identity is sufficiently maintained to allow recognition 
by complementary binding partners, such as antibodies. However, 
immobilization of functional proteins such as enzymes, antibodies, 
specific binding proteins, etc. must be performed in a benign manner in 
order to preserve their binding or reactive properties. 
There is a need for high refractive index particle reagents for use in 
specific binding assays, especially immunoassays, that provide reactive 
groups for covalent protein attachment under mild conditions that preserve 
protein functional or antigenic integrity. This is especially critical 
when proteins of interest are scarce or have multiple subunits with active 
binding regions which might be rendered less active by disassociation 
under severe coupling conditions. Having autoreactive functional groups 
capable of coupling to proteins under mild conditions and which remain 
stable and active upon storage in aqueous media and in the presence of 
other reagents such as detergents and salts, would minimize reagent 
manipulations and maximize product reproducibility from separate 
syntheses. A core-shell particle having stable, autoreactive shell 
functional groups that covalently immobilize proteins under mild coupling 
conditions, would be highly desireable to provide useful reagents for 
light scattering specific binding assays. 
DISCLOSURE OF THE INVENTION 
The particle reagent of this invention has a high refractive index and 
consists essentially of: 
A. a polymer particle having an inner core and an outer shell wherein the 
inner core is a polymer having a refractive index of not less than 1.54 as 
measured at the wavelength of the sodium D line and wherein the outer 
shell is a polymer of 
(1) at least five parts by weight of the outer shell of an ethylenically 
unsaturated monomer having a haloalkyl functional group capable of 
reacting with a compound of biological interest, its antigen or its 
antibody, selected from the group consisting of 
##STR1## 
wherein X is Cl or Br and R is H, CH.sub.3, or C.sub.2 H.sub.5, (2) 
optionally other ethylenically unsaturated monomers in amounts not 
resulting in the formation of water soluble polymer particles, and 
(3) not more than 10 parts by weight of the outer shell of the residual 
monomers of the inner core; 
said outer shell being formed by polymerization in the presence of said 
inner core; and wherein said polymer particle has an approximate diameter 
range of 0.01-1.0 .mu.m, a 5-100% surface coverage by a monomolecular 
layer of anionic surfactant, and is covalently attached to 
B. a compound of biologicl interest, its antigen or its antibody. 
The method of this invention for the detection and measurement of compounds 
of biological interest (which are intended to include the analogs of such 
compounds), their antibodies or antigens utilizes the particle reagents as 
described above. 
DESCRIPTION OF THE INVENTION 
This invention relates to novel particle reagents having high 
immunoreactivity and which are useful in high sensitivity light scattering 
immunoassays. These particle reagents are an improvement over the 
core-shell particles disclosed in commonly assigned U.S. Pat. No. 
4,401,765, issued Aug. 30, 1983, incorporated herein by reference, in that 
they provide reactive halide groups on the surface of core-shell particles 
that impart several advantages. 
The activated shell monomers having halide leaving groups such as chloride 
and bromide are reactive with protein amine and amide groups. There is no 
requirement for a separate, preliminary activating step to render the 
particle reagents reactive with desired biological materials. This 
convenience allows fewer handling and transfer steps in the preparation of 
particle reagents. In addition, the halide leaving groups on the particle 
shell are sufficiently stable to hydrolysis under various conditions of 
pH, ionic strength, temperature, etc. during aqueous storage and handling 
that a significant proportion of the reactive groups are retained. This 
reactive group stability provides greater control over covalent 
immobilization of materials of biological interest. For purposes of 
brevity, materials of biological interest include their antigens and 
antibodies. Further, the hydrolytic stability of the halide groups under 
particle synthetic conditions allows the preparation of well defined 
reagents having a known proportion of reactive groups. For example, a 
chloromethylstyrene monomer can be polymerized onto a polymer core in any 
proportion to other halide or non-halide containing monomers, to produce a 
shell having from 5 to 100% chloromethylstyrene monomers. The original 
proportion of reactive groups can be maintained in an aqueous environment 
for a sufficient period to allow the reproducible synthesis of core-shell 
polymer particles that can be later reacted with proteins in a 
reproducible manner. 
Shell compositions that comprise from about 10 to 100% haloalkyl monomers 
are generally more useful and preferred in making the particle reagents of 
this invention. Shell compositions comprising less than 10% haloalkyl 
monomers and other ethylenically unsaturated monomers, while capable of 
immobilizing biological materials, do so in amounts too low to be useful. 
Immunoassays performed using particle reagents having shell compositions 
with less than 10% haloalkyl monomers are expected to be less sensitive 
than those using particle reagents derived from shell compositions having 
greater than 10% haloalkyl monomer. However, in those assay circumstances 
where a reduced level of immobilized biological material may be desired 
such as to encourage cross-linking even where only low levels of divalent 
antibody are available, the particle reagents having shells with less than 
10% but at least 5% haloalkyl monomers may be useful. 
The advantages of the core-shell particle configuration disclosed in U.S. 
Pat. No. 4,401,765 are maintained in the present invention. The light 
scattering properties of particle suspensiions depend on several 
variables, most importantly the particle size, the refractive indices of 
the core and the suspension medium, and the wavelength of light used for 
measurement. Thus, the selection of core material, particle size, and 
wave-length of detection of the agglutination reaction are all important 
factors in optimizing assay sensitivity. These factors can be determined 
by the type of light scattering detection means used. 
During visual observation of the agglutination reaction, a broad band of 
wavelengths, between approximately 400 and 600 nm, can be utilized. Since 
the light scattering response varies over this wavelength range, the 
visual observation results in an averaging of the effects of many 
wavelengths which is less sensitive than choosing the optimum wavelength 
for a given particle size and refractive index. For particles whih are 
small compared to the wavelength of light, the scattering increases with 
the inverse 4th power of the wavelength and the magnitude is dependent 
upon the refractive index, when the wavelength of light approaches an 
adsorption band of the particle, there is an increase of refractive index 
and thus the light scattering properties are sensitive also to the optical 
dispersion of the scattering element and the wavelength functionality may 
exceed the 4th power. 
For the turbidimetric detection of particle size change at a given 
wavelength or measurement it is imperative that the particle size and 
refractive index be chosen with care since the turbidimetric signal goes 
through a maximum, producing a double-valued response with little or no 
sensitivity at the peak. In addition, the slope sensitivity is greater on 
the small particle size side of the peak than on the large and it 
increases with increasing refractive index ratio of particle to medium. 
For these reasons, small particles of high refractive index with short 
wavelength detection are preferred for high sensitivity. There is a 
practical limit in the ultraviolet region for measurement of samples in 
serum because of light adsorption by proteins and other components. Thus, 
convenient wavelengths are those in excess of approximately 320 nm. 
Shorter wavelengths, such as 340 nm, give larger signal differences than 
longer wavelengths, such as 400 nm. In general, particle size range of 
0.01-1.0 .mu.m can be utilized in the particle reagent of this invention. 
For nephelometric detection, the optimum sensitivity can depend not only on 
particle size and wavelength, but also on the angle of measurement. 
Nephelometry refers to the measurement of the light scattered at an angle 
from the incident beam. The size of the particles for optimum sensitivity 
will have an angular dependence as well as a wavelength dependence. 
Other types of scattering measurements of the agglutination reaction 
include particle counting, quasi-elastic light scattering, autocorrelation 
spectroscopy, and measurements of the dissymmetry or the polarization of 
the particles. These types of measurements provide different constraints 
for the particle reagents. 
In all types of measurements, however, the higher the refractive index of 
the particles at the wavelength of choice, the higher the light scattering 
signal. 
A preferred way of measurement of immunological reactions utilizing the 
particle reagents of this invention is by turbidity since no special 
equipment is required other than a spectrophotometer which is generally 
available in clinical laboratories. The spectrophotometer measures 
increased absorbance which is due to the increasing particle size 
resulting from the agglutination reaction. This increased absorbance is a 
direct measure of the agglutination caused by the analyte or an indirect 
measure of the agglutination inhibition caused by the analyte. To optimize 
the turbidity change which occurs during agglutination, it is important to 
select the particle size with care. 
During the agglutination reaction, the effective particle size increases. 
For sensitive measurements it is, therefore, important to choose the 
wavelength at which the signal change for a given particle size change is 
optimal. 
Because of the importance of the refractive index for turbidimetric 
detection of the agglutination reaction, core materials are restricted to 
those which will produce acceptable signal changes for the desired assay 
sensitivity. For analytes in high concentrations (.mu.g/mL range), the 
choice is not critical, but for analytes in the nanogram/mL range, 
particles having high refractive index are necessary. Thus, core polymers 
with high aromaticity and high atomic weight substituents are preferred 
over aliphatic polymers and, in general, polymers of high refractive 
indices are preferred over polymers with lower refractive indices. 
The inner core of the polymer particles can be selected from a large group 
of materials with high refractive index. Preferred are those materials 
which can be prepared by emulsion polymerization in a manner so that the 
final particle size is controllable and is substantially uniform. Polymers 
utilized in the inner core of the polymer particles have refractive 
indices greater than 1.54 (at the Na D line, 569 nm) and are listed in 
Table 1. Since the refractive index is a function of wavelength, the 
scattering properties will be dependent upon the wavelength of 
measurement. In general, the refractive index is greater at shorter 
wavelengths. 
TABLE 1 
______________________________________ 
REFRACTIVE INDICES OF POLYMERS 
Polymer n.sub.D 
______________________________________ 
Cellulose 1.54 
Poly(vinyl chloride) 1.54-1.55 
Urea-formaldehyde resin 1.54-1.56 
Poly(sec-butyl .alpha.-bromoacrylate) 
1.542 
Poly(cyclohexyl .alpha.-bromoacrylate) 
1.542 
Poly(2-bromoethyl methacrylate) 
1.5426 
Poly(dihydroabietic acid) 1.544 
Poly(abietic acid) 1.546 
Poly(ethylmercaptyl methacrylate) 
1.547 
Poly(N--allyl methacrylamide) 
1.5476 
Poly(1-phenylethyl methacrylate) 
1.5487 
Poly(vinylfuran) 1.55 
Poly(2-vinyltetrahydrofuran) 
1.55 
Poly(vinyl chloride) + 40% 
1.55 
tricresyl phosphate 
Epoxy resins 1.55-1.60 
Poly(p-methoxybenzyl methacrylate) 
1.552 
Poly(isopropyl methacrylate) 
1.552 
Poly(p-isopropylstyrene) 1.554 
Poly(chloroprene) 1.554-1.55 
Poly(oxyethylene)-.alpha.-benzoate-.alpha.- 
1.555 
methacrylate) 
Poly(p,p'-xylylenyl dimeth- 
1.5559 
acrylate) 
Poly(1-phenylallyl methacrylate) 
1.5573 
Poly(p-cyclohexylphenyl 1.5575 
methacrylate) 
Poly(2-phenylethyl methacrylate) 
1.5592 
Poly(oxycarbonyloxy-1,4- 1.5602 
phenylene-1-propyl-butylidene- 
1,4-phenylene) 
poly[1-(o-chlorophenyl)ethyl 
1.5624 
methacrylate] 
Poly(styrene-co-maleic anhydride) 
1.564 
Poly(1-phenylcyclohexyl 1.5645 
methacrylate) 
Poly(oxycarboxyloxy-1,4-phenylene- 
1.5671 
1,3-dimethylbutylidene-1,4- 
phenylene) 
Poly(methyl .alpha.-bromoacrylate) 
1.5672 
Poly(benzyl methacrylate) 1.5680 
Poly[2-(phenylsulfonyl)ethyl 
1.5682 
methacrylate] 
Poly(m-cresyl methacrylate) 
1.5683 
Poly(styrene-co-acrylonitrile) 
1.57 
(ca. 75/25) 1.57 
Poly(oxycarbonyloxy-1,4-phenyl- 
1.5702 
eneisobutylidene-1,4-phenylene) 
Poly(o-methoxyphenyl methacrylate) 
1.5705 
Poly(phenyl methacrylate) 1.5706 
Poly(o-cresyl methacrylate) 
1.5707 
Poly(diallyl phthalate) 1.572 
Poly(2,3-dibromopropyl 1.5739 
methacrylate) 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.5745 
1-methylbutylidene-1,4-phenylene) 
Poly(oxy-2,6-dimethylphenylene) 
1.575 
Poly(oxyethyleneoxyterephthalate) 
1.575 
Poly(vinyl benzoate) 1.5775 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.5792 
butylidene-1,4-phenylene) 
Poly(1,2-diphenylethyl 1.5816 
methacrylate) 
Poly(o-chlorobenzyl methacrylate) 
1.5823 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.5827 
sec-butylidene-1,4-phenylene) 
Poly(oxypentaerythritoloxyphthalate) 
1.584 
Poly(m-nitrobenzyl methacrylate) 
1.5845 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.5850 
isopropylidene-1,4-phenylene) 
Poly(N--2-phenylethyl methacrylamide) 
1.5857 
Poly(4-methoxy-2-methylstyrene) 
1.5868 
Poly(o-methylstyrene) 1.5874 
Poly(styrene) 1.59-1.592 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.5900 
cyclohexylidene-1,4-phenylene) 
Poly(o-methoxystyrene) 1.5932 
Poly(diphenylmethyl methacrylate) 
1.5933 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.5937 
ethylidene-1,4-phenylene) 
Poly(p-bromophenyl methacrylate) 
1.5964 
Poly(N--benzyl methacrylamide) 
1.5965 
Poly(p-methoxystyrene) 1.5967 
Hard rubber (32% S) 1.6 
Poly(vinylidene chloride) 1.60-1.63 
Poly(sulfides) 1.6-1.7 
Poly(o-chlorodiphenylmethyl 
1.6040 
methacrylate) 
Poly[oxycarbonyloxy-1,4-(2,6- 
1.6056 
dichloro)phenylene-isopropyli- 
dene-1,4-(2,6-dichloro)phenylene)] 
Poly[oxycarbonyloxybis 1,4-(3,5- 
1.6056 
dichlorophenylene)] 
Poly(pentachlorophenyl methacrylate) 
1.608 
Poly(o-chlorostyrene) 1.6098 
Poly(phenyl .alpha.-bromoacrylate) 
1.612 
Poly(p-divinylbenzene) 1.6150 
Poly(N--vinylphthalimide) 1.6200 
Poly(2,6-dichlorostyrene) 1.6248 
Poly(.alpha.-naphthyl methacrylate) 
1.6298 
Poly(.alpha.-naphthyl carbinyl 
1.63 
methacrylate) 
Poly(sulfone) 1.633 
Poly(2-vinylthiophene) 1.6376 
Poly(.alpha.-naphthyl methacrylate) 
1.6410 
Poly(oxycarbonyloxy-1,4-phenylene- 
1.6539 
diphenylmethylene-1,4-phenylene) 
Poly(vinyl phenyl sulfide) 
1.6568 
Butylphenol-formaldehyde resin 
1.66 
Urea-thiourea-formaldehyde resin 
1.660 
Poly(vinylnaphthalene) 1.6818 
Poly(vinylcarbazole) 1.683 
Naphthalene-formaldehyde resin 
1.696 
Phenol-formaldehyde resin 1.70 
Poly(pentabromophenyl methacrylate) 
1.71 
______________________________________ 
Not all of the polymers listed above can be utilized as the inner core for 
the particle reagents of this invention since there are additional 
criteria to be applied to the selection of core monomer materials. 
Cellulose, for example, is not readily prepared as uniform particle size 
spheres. Condensation polymers are also not useful since the 
polymerization process does not lead to spherical particles of the type 
which can be obtained by emulsion polymerization. Some thermoplastic 
polymers such as poly(oxyethylene-oxyterephthalate) and some thermosetting 
resins of the urea-formaldehyde type are not suitable. 
The monomers of interest are those which contain vinyl or allyl groups in 
addition to substituents such as halides, aromatic, heterocyclic, 
unsaturated or carbocyclic groups which impart high refractivity. 
Polymer particles useful for the preparation of the particle reagents of 
this invention can be prepared preferentially by emulsion polymerization. 
Staged emulsion polymerization can lead to a core/shell polymer 
approximating the desired refractive index of not less than n.sub.D =1.54. 
To obtain a polymer of desired refractive index, it is preferred that the 
shell polymer not exceed approximately 10 parts by weight of the polymer 
particle. 
A convenient way to control particle size of the polymer particles is to 
first prepare a seed emulsion whose size can be controlled by the amount 
of surfactant used. After preparation of the seed emulsion, additional 
monomer and surfactants can be added at a controlled rate to increase the 
size of the particles in the seed emulsion. 
It is preferable to carry the conversion of the core monomer(s) to 
substantial completion so that the shell polymer be a homopolymer or a 
copolymer of known composition rather than a copolymer of unknown 
composition. Conversions in excess of 98% can be attained by increasing 
the temperature of the core emulsion to approximately 98.degree. C. at the 
end of the polymerization. To further reduce the probability of producing 
particles whose surface is a copolymer of unknown composition, the shell 
monomer can be added gradually rather than batchwise. In such a manner, 
the residual core monomer(s) can be consumed during the early stages of 
the shell polymer formation. 
The attachment of the shell polymer to the core can be accomplished by 
graft polymerization of the functional monomer to the residual 
ethylenically unsaturated groups in the core polymer or the functional 
monomer can be polymerized around the core to produce a contiguous shell. 
Preferred monomers include chloromethylstyrene and bromomethylstyrene, the 
most preferred being chloromethylstyrene. 
The outer shell of the polymer particle can be prepared from a range of 
haloalkyl monomers having functional groups capable of reacting with 
desired biological materials, especially proteins. The outer shell can be 
a homopolymer of haloalkyl monomers or copolymers of such haloalkyl 
monomers and other ethylenically unsaturated monomers in amounts not 
resulting in the formation of water soluble polymer particles. The 
selection of appropriate outer shell monomers will depend largely upon 
their intended use. However, the copolymer compositions which include 
active haloalkyl monomers and other ethylenically unsaturated monomers can 
be prepared by controlled synthetic procedures to produce particle 
reagents having defined amounts of shell reactive groups. 
The relative amount of the reactive haloalkyl groups in the shell affects 
the activity of the covalently attached biological material in a manner 
not clearly understood. The biological reactivity (such as antibody 
binding, enzyme activity, etc.) of the material covalently bound to the 
particle shell exhibits an optimum, when evaluated by functional assays 
such as immunoassays, enzyme-substrate reactions, etc., that is related to 
the amount of active halide in the shell. Therefore, control of the 
polymerization reactions which produce the shell polymers is important to 
insure that the concentration of reactive halide groups in the shell be 
reproducible. This control can be achieved by sequential synthesis of core 
and shell polymers in a manner that insures essentially complete core 
polymerization prior to shell polymerization. The outer shell should not 
contain more than 10 parts, preferably not more than 5 parts, and even 
more preferably not more than 2 parts, by weight of the outer shell of the 
monomers of the inner core. These limitations on inner core monomers are 
meant to apply only to the residual monomers not polymerized during the 
preparation of the inner core. Regulation of polymerization times, 
temperatures, surfactant levels, monomer concentrations, etc. allows the 
synthetic control required to produce defined shell compositions on 
polymer cores that result in polymer particles useful in the present 
invention. 
The amount of active haloalkyl monomer polymerized into the particle shell 
has been unexpectedly found to influence the biological activity of 
covalently bound proteins such as antibodies. Lower amounts of haloalkyl 
monomers in the range of 10 to 30% (w/w) relative to other ethylenically 
unsaturated, inactive monomers in the particle shell, have been found to 
provide better specific binding activity of particle reagents having 
antibody covalently bound to them than in antibody particle reagents 
having shells polymerized with greater than about 30% (w/w) haloalkyl 
monomer. In contrast, proteins such a antigens, protein carriers of 
haptens, peptide hormones, etc. can be covalently immobilized onto 
particle shells composed of as much as 100% chloromethylstyrene without 
significant loss of identity to complementary binding agents. These 
observations suggest that immobilization of biologically functional 
materials such as antibodies, enzymes, hormone receptors, etc. that rely 
upon appropriate stereochemical associations of subunits to provide active 
sites should be performed using particles with shells having lower 
relative concentrations of active haloalkyl monomer in the range of 10 to 
30% (w/w). 
A preferred way of carrying out the shell polymerization process is in the 
presence of anionic surfactants (such as sodium dodecyl sulfate, lithium 
dodecyl sulfate, GAFAC.RTM. RE610, a mixture of octyl and nonylphenyl 
ethers of polyoxyethylene terminated with a phosphate group, etc., 
preferably sodium dodecyl sulfate). Alternatively, anionic surfactant can 
be added after this process to create and maintain a net negative charge 
on the shell surface. This charged environment provides for the adsorption 
of biological materials, especially proteins, onto the hydrophobic 
particle shell without creating deleterious conditions that would promote 
denaturation or other irreversible changes that could severely diminish 
structural integrity or biological activity. The anionic surfactant also 
provides conditions that favor maintaining monodisperse particle 
suspensions during particle synthesis as well as after protein adsorption 
and covalent attachment to reactive shell groups. It has been found that 
anionic surfactant are useful in a concentration range such that at least 
5% of the latex surface is covered with a monomolecular layer of 
surfactant. 
Attempts to use cationic or, surprisingly, even nonionic surfactants to 
prepare particle reagents having biological materials covalently attached 
to them resulted in aggregated or inactive particle reagents. These 
reagents were unsuited for use in immunoassays when antibody or 
proteinaceous antigens were covalently bound to particle surfaces in the 
presence of cationic surfactants because of poor light scattering 
efficiency of the aggregated particle reagent. Use of a nonionic 
surfactant, such as Triton X-100, in place of cationic surfactants, 
produced a suitably dispersed particle preparation. However, attampts to 
attach proteins covalently to the haloalkyl groups on the particle shell 
failed to produce particles with any significant activity. Although the 
particle reagent remained sufficiently dispersed in the liquid medium, no 
useful immunoassays could be carried out with the reagent. It appears, 
therefore, that the use of anionic surfactants is an unexpected 
requirement for the preparation and use of the particle reagents of the 
present invention. 
The present invention is further concerned with an immunologically active, 
stable particle reagent for use in sensitive light scattering immunoassays 
for detecting and measuring compounds of biological interest. These assays 
are contemplated to be used to detect and measure a wide variety of 
substances in biological fluids and cell and tissue extracts for which an 
immunological counter reactant can be produced. The compounds of 
biological interest include serum and plasma proteins, salivary, urinary 
or milk proteins, drugs, vitamins, horomones, enzymes, antibodies, 
polysaccharides, bacteria, protozoa, fungi, viruses, cell and tissue 
antigens and other blood cell or blood fluid substances. Of special 
interest are those substances for which a quantitative determination is 
required for the assessment of disease state. 
The amount of haloalkyl monomer to be incorporated into the shell portion 
of the particle reagent of this invention can be dictated by the type and 
amount of biological material to be immobilized. For example, to 
immobilize antibody, a polymer particle having a shell containing from 
about 10 to 30% (w/w) chloromethylstyrene can be mixed with the 
appropriate amount of an anionic surfactant, such as SDS. The amount of 
surfactant can be calculated from the known (precalculated) particle 
surface area and from known surfactant parameters [from Polymer Handbook, 
Brandrup and Immergent (eds.), J. Wiley & Sons, 2.sup.nd Edition (1975) 
Section II, p. 485]. It is preferable to provide sufficient anionic 
surfactant to cover between 40 and 100% of the particle surface with a 
surfactant monolayer. Protein can be added in molar excess over active 
shell halides in the range of a 5:1 to 50:1 molar ratio. After mixing in a 
buffered solution to insure complete exposure of particle surface 
functional groups to proteins, the mixture is allowed to incubate over a 
period of from 0.5 to 10 hours, preferably 0.5 to 3 hours, at a 
temperature in the range of 25.degree. to 40.degree. C. When incubation is 
carried out at 4.degree. C., it should be extended over several days to 
achieve adequate covalent attachment of protein to the particles. After 
sufficient time has elapsed to allow covalent attachment, a separation of 
particle reagent from the suspending buffered medium is effected, usually 
by centrifugation, although filtration, gravitational settling, etc. would 
suffice to allow the removal of unbound protein. 
In the preparation of antigen particle reagent, several washings of the 
particle reagent are performed with buffered solution, usually containing 
anionic surfactant. For example, a glycine or phosphate buffer can be used 
containing a surfactant such as GAFAC.RTM. RE610, in the range of from 0.1 
to 0.5% (w/v). The washings are carried out by sequential suspensions and 
separations of the particle reagent and removal of supernatants. This 
washing process is repeated a sufficient number of times, usually three to 
five times, until the supernatant solution contains substantially no 
protein. 
The particle reagent so produced can be tested for immunoreactivity by 
turbidimetric assay with the binding partner complementary to the partner 
covalently linked to the polymer particle. The particle reagents of this 
invention exhibit a high degree of immunoreactivity, indicating that 
immunoreactants have been covalently linked to shell-core polymer 
particles in the presence of anionic surfactants, in a manner sufficiently 
benign to preserve a significant proportion of their original activity. 
In other instances, such as immobilization of an antibody on polymer 
particles, where the antibody serves as a specific binding agent for 
materials of biological interest such as C-Reactive Protein, anionic 
surfactant does not have to be incorporated in the wash buffers. It is 
preferred that the level of anionic surfactant used in the synthesis of 
the core-shell polymer particle, prior to protein attachment, be 
sufficient to provide a calculated particle surface coverage of between 
about 40 and 70%. It has been found that antibody particle reagents are 
more active when the anionic surfactant is introduced during the 
polymerization process, rather than being added during protein attachment 
or in the wash buffers. When this method is used to bind antibodies to the 
haloalkyl shell groups, it is believed that sufficient anionic surfactant 
is associated with the particle surface during protein attachment to 
provide optimal conditions for maintenance of the appropriate particle 
dispersion, without deleteriously affecting antibody activity. 
Antibodies appropriate for use in the present invention can include 
antibodies capable of binding antigen or hapten, are selected from any 
class or subclass of antibody, and can be polyclonal or monoclonal. 
Fragments of antibodies, such as F(ab').sub.2, F(ab'), and F(ab), can also 
be used. When monovalent antibody is used, it should be immobilized onto 
polymer particles to function as an agglutinating agent or when it is the 
compound of biological interest. 
Immunoassays utilizing the particle reagents of this invention can be 
designed in a variety of ways depending on the type of analyte and on the 
required sensitivity. For example, for analytes in relatively high 
concentrations such as certain serum proteins, appropriate antibody 
particle reagents can be used in direct particle enhanced turbidimetric 
immuno-precipitation techniques. 
The inhibition immunoassay method of this invention also requires, in 
addition to a particle reagent, a bi- or multi-functional agent, 
hereinafter referred to to as an agglutinating agent, to cause the 
agglutination of the particle reagent. It is this agglutination which can 
be initiated by the compound of biological interest. The agglutinating 
agent can be an antibody to the compound of biological interest or another 
particle reagent based on a polymer particle, as described above, 
covalently attached to an antibody of the compound of biological interest. 
The agglutinating agent can also be a multivalent conjugate of the 
compound of biological interest and a protein. Such a conjugate may be 
utilized in situations where the particle reagent utilized in the method 
of this invention contains a covalently attached antibody of the compound 
of biological interest. 
For the measurement of haptens such as drugs, several different assay 
configurations can be utilized. In one such configuration, antigenic 
particle reagents can be prepared by attaching a hapten-protein conjugate 
to the polymer particle. The inhibition of the reaction between these 
particle reagents and the appropriate antibodies by the hapten of 
biological interest is determined. The reaction can be performed by direct 
competition for the antibody between the particle reagent and the sample 
hapten or by sequential reaction of the hapten with antibody followed by 
addition of the particle reagent. 
Another assay configuration for haptens can utilize antibody particle 
reagent wherein the agglutination of the antibody particle reagents with 
soluble multi-haptenic protein conjugates is inhibited by the analyte 
(hapten). Such an assay can also be performed in a competitive or 
sequential mode. In yet another assay, both antibody and multi-haptenic 
particle reagents can be present, of the same or differing sizes, and the 
inhibition reaction by haptens can be performed in a competitive or 
sequential mode. 
The agglutination reaction, in general, can be accelerated by the presence 
of an agglutinating accelerator. Such an accelerator can be a polyethylene 
glycol or an anionic surfactant such as sodium dodecyl sulfate. 
The following examples are illustrative of the invention.

EXAMPLES 
Example 1 
Comparison of Particles Having Polychloromethylstyrene (PCMS) Shell and 
Particles Having Polyglycidyl Methacrylate (PGMA) Shell for Immobilization 
of Fibrinogen Degradation Products (FDP) 
(A) Preparation of Polystyrene/Polyvinylnaphthalene/Polychloromethylstyrene 
Core-Shell Polymer Particles 
(i) Polystyrene Seed Emulsion Polymerization 
A polystyrene seed emulsion was prepared at room temperature (20.degree. 
C.) by adding to a 4 L-Ehrlenmeyer flask, under a nitrogen atmosphere, 2.5 
L deionized water, 400 g Dupanol WAQE (30% solution of SDS, available from 
E. I. du Pont de Nemours and Co.), 50 mL styrene, 20 g sodium 
metabisulfite, 200 mL of a 350 mg/mL potassium persulfate solution, and 
125 mL of a ferrous sulfate solution (0.6 g of ferrous sulfate 
heptahydrate and 0.25 g of sulfuric acid dissolved in 500 mL of 
nitrogen-purged, deionized water). After 10 minutes, 25 g of Aerosol 
OT-100 dissolved in 376 mL of styrene was added at a rate of 30 mL/min. 
The mixture was stirred overnight. A sample of the product, diluted 1:100, 
had an optical density of 0.171 when measured at 340 nm. 
(ii) 2-Vinylnaphthalene Core Polymerization 
Latex polymerization to prepare polyvinylnaphthalene was carried out in a 
250-mL round-bottomed flask equipped with a magnetic stirrer and a reflux 
condenser. 1.8 mL of the polystrene seed emulsion prepared in Example 1Ai 
above was added to 98 mL of water, and the mixture was heated to 
95.degree. C. This mixture was then added to 11.7 g of 2-vinylnaphthalene 
(2-VN, purified by sublimation and chromatography on basic alumina in a 
dichloromethane solution), 300 mg of sodium bicarbonate and 90 mg of 
potassium persulfate. As soon as the 2-vinylnaphthalene had melted, 4 mL 
of a 10% sodium dodecyl sulfate solution was added at a rate of 0.3 
mL/min. One hour after the beginning of the SDS feed, the polymerization 
was conplete. The optical density of the product measured at 340 nm was 
0.155 after diluting 1:5000 in water. The number average particle size was 
determined to be 0.074 .mu.m by electron microscopy. The conversion of 
monomer to polymer was found to be 99.6% by gas chromatographic 
determination of the residual monomer. 
(iii) Preparation of 2-VN/CMS Core-Shell Polymer 
104 mL of the polyvinylnaphthalene latex prepared in Example 1Aii was 
preheated in the same apparatus as utilized above to about 95.degree. C. 
and 100 mg of potassium persulfate and 2.06 mL of chloromethylstyrene 
(CMS, Polysciences Co.; mixed isomers) were added. After 1 hour, the 
mixture was cooled. The conversion of chloromethylstyrene to polymer was 
found to be 93% complete. The optical density of a 1:5000 diluted sample 
was measured at 340 nm and found to be 0.183. 
(B) Preparation of Polyvinylnaphthalene/Polyglycidyl Methacrylate 
Core-Shell Polymer Particles 
(i) Polystyrene/polyvinylnaphthalene latex was prepared as in Example 1Aii. 
(ii) Glycidylmethacrylate Shell Polymerization 
The procedure used was the same as in Example 1Aiii, except that the 
polymerization time was 20 minutes, instead of 1 hour, and glycidyl 
methacrylate was substituted for chloromethylstyrene. The conversion of 
monomer to polymer was determined to be 99.0%. The final optical density 
of a 1:5000 diluted sample was 0.154 when measured at a wavelength of 340 
nm. 
(C) Immobilization of Fibrinogen Degradation Products (FDP) 
1 mL of each final latex from Examples 1A and 1B in separate but identical 
procedures, was first mixed with a volume (specified below) of a 10% 
sodium dodecyl sulfate (SDS) solution and 4 mL of a 15 mM phosphate 
buffer, pH 7.5. This suspension was then added to a solution of 10 mg of 
FDP [prepared by the method of Matsushima et al., Thrombosis Research, 
Volume 27, 111-115 (1982)] in 5 mL of 15 mM phosphate buffer (as above). 
The pH of the mixture was adjusted to 7.8 using 0.1M sodium hydroxide, and 
incubated for the time and temperature indicated in Table 1. 
After the specified incubation time, each latex was centrifuged at 20,000 
RPM in a Sorvall.RTM. model RC-5B centrifuge (a registered trademark of E. 
I. du Pont de Nemours and Co.). The supernatant was decanted from the 
pellet of particles, and the pellet resuspended in a 0.1% solution of 
GAFAC.RTM. RE610 (an anionic detergent available from GAF Corporation) in 
15 mM phosphate buffer, pH 7.5, by sonication for 3 minutes with a Heat 
Systems Ultrasonics model 225R sonicator. The suspension was again 
centrifuged and resuspended as above. Finally, it was centrifuged a third 
time, the supernatant decanted, and the particle pellet dried under 
vacuum. 
The dried pellet was then analyzed for nitrogen content using the Kjeldahl 
method. 
The results for the two types of latex are summarized below in Table 2. 
TABLE 2 
______________________________________ 
Covalent Attachment of FDP to Latex Particles 
Volume of Incubation 
Incubation 
Nitrogen 
Type of 
10% SDS Time Temperature 
Content 
Shell (.mu.L) (hour) (.degree.C.) 
(%, Average) 
______________________________________ 
PCMS 100 0.5 4 0.005 
100 0.5* 4 0.385 
100 24 4 0.175 
100 24 37 0.335 
PGMA 100 0.5 4 0.00 
100 0.5* 4 0.005 
100 24 4 0.00 
100 24 37 0.005 
0 0.5 4 0.125 
0 0.5* 4 0.685 
0 24 4 0.400 
0 24 37 0.710 
______________________________________ 
*Samples were centrifuged only once, then decanted, and the pellet dried 
and analyzed. From the data one can conclude that a single centrifugation 
results in incomplete removal of unbound FDP. 
The data show that during polymer particle-protein interactions in the 
presence of SDS, even after two GAFAC.RTM. RE610 washings, FDP remains 
associated with the PCMS shell but not significantly associated with the 
PGMA shell. These results indicate that FDP is covalently linked to PCMS 
particles under the incubation conditions specified, but not to PGMA 
particles. (The covalent linkage of FDP with PCMS latex even at an 
incubation temperature of 4.degree. C. was an added advantage since low 
temperatures are generally better suited to retention of biological 
activity of proteins than elevated temperatures.) In the absence of SDS, 
protein (FDP) did associate with PGMA but the particle reagent so produced 
was highly aggregated and was unsuitable for use in immunoassays for FDP. 
The role played by the anionic surfactant in providing conditions to permit 
the production of useful PCMS-based particle reagents was surprising. In 
addition, the successful use of cationic and nonionic surfactants in 
association with a core-shell particle having haloalkyl functional groups 
(U.S. Pat. No. 4,017,442) provided no incentive to use anionic 
surfactants. In contrast, the failure to produce acceptable PGMA-based 
particle reagent in the presence of added anionic surfactant was also 
surprising in that U.S. Pat. No. 4,401,765 disclosed the use of anionic 
surfactants to produce particle reagents having protein covalently linked 
through the epoxy functional group of PGMA. 
Example 2 
Immobilization of Fibrinogen on Particles Having Polychloromethylstyrene 
Shell and Particles Having Polyglycidyl Methacrylate Shell 
The same procedures were used as in Example 1 except that fibrinogen, and 
not fibrinogen degradation products, was reacted with polymer particles at 
4.9 mg/mL fibrinogen. Fifty percent higher SDS levels were also added 
during particle reagent synthesis. Fibrinogen was obtained from Helena 
Laboratories (Lot #8266781). The results are presented in Table 3. 
TABLE 3 
______________________________________ 
Covalent Attachment of Fibrinogen to Latex Particles 
Volume of Incubation 
Incubation 
Nitrogen 
Type of 
10% SDS Time Temperature 
Content 
Shell (.mu.L) (hour) (.degree.C.) 
(%, Average) 
______________________________________ 
PCMS 150 .5 4 0.305 
150 .5 4* 0.505 
150 24 4 0.420 
150 24 37 0.370 
PGMA 150 .5 4 0.01 
150 .5 4* 0.01 
150 24 4 0.00 
150 24 37 0.00 
0 .5 4 .infin. 
0 .5 4* 1.35 
0 24 4 .infin. 
0 24 37 .infin. 
______________________________________ 
*Samples were centrifuged only once, then decanted, and the pellet dried 
and analyzed. From the data one can conclude that a single centrifugation 
results in incomplete removal of unbound fibrinogen. 
.infin. Particle pellets could not be resuspended after the first 
centrifugation 
The data show results similar to those obtained in Example 1. Fibrinogen is 
associated with PCMS in the presence of SDS at both 4.degree. and 
37.degree. C., but not with PGMA latex. When no additional SDS was added 
during the procedure, the PGMA particle reagents were so highly aggregated 
after the first centrifugation that they could not be resuspended for 
further processing. 
Example 3 
Immobilization of Rabbit IgG Protein on Particles Having 
Polychloromethylstyrene Shell and Particles Having Polyglycidyl 
Methacrylate Shell 
Rabbit IgG (Cappel Diagnostics; chromatography purified Lot #20872) was 
attached to latex polymer particles, prepared in Examples 1A and 1B, using 
the same protocol as was used with FDP in Example 1. The data for 
comparison of immobilized protein on PCMS and PGMA latices are summarized 
in Table 4. 
TABLE 4 
______________________________________ 
Covalent Attachment of Rabbit IgG to Latex Particles 
Volume of Incubation 
Incubation 
Nitrogen 
Type of 
10% SDS Time Temperature 
Content 
Shell (.mu.L) (hour) (.degree.C.) 
(%, Average) 
______________________________________ 
PCMS 100 0.5 4 0.145 
100 0.5* 4 0.685 
100 24 4 0.245 
100 24 37 0.470 
PGMA 100 0.5 4 0.00 
100 0.5* 4 0.20 
100 24 4 0.005 
100 24 37 0.01 
______________________________________ 
*Samples were centrifuged only once, then decanted, and the pellet dried 
and analyzed. The data indicate incomplete removal of unbound IgG. 
The data show that in the presence of added SDS, only PCMS-shell polymer 
particle of this invention was suitable for immobilization of rabbit IgG. 
Example 4 
Measurement of Fibrinogen Degradation Products 
An automated turbidimetric inhibition immunoassay was performed at 
37.degree. C. on the aca.RTM. discrete clinical analyzer (a registered 
trademark of E. I. du Pont de Nemours & Company). Assay standards were 
prepared by adding fibrinogen degradation products (FDP) to normal human 
serum. Fibrinogen degradation products were prepared by degrading 200 mg 
of purified fibrinogen with 25 CU (casein units) of plasminogen that was 
activated by 500 U of streptokinase at 37.degree. C. After 1 hour, the 
reaction was terminated by the addition of soybean trypsin inhibitor to a 
final concentration of 1 mg/mL. Greater than 95% fibrinogen degrdation was 
established by comparison of the starting fibrinogen material with the 
products in the final degradation mixture using a standard 
SDS-polyacrylamide gel electrophoresis analytical procedure. This FDP 
solution was then serially diluted to provide the FDP levels calculated 
from the starting fibrinogen level of 200 mg by assuming substantially 
complete degradation; see Table 5. The zero level standard was 5% (w/v) 
human serum albumin in water. 
A 20-.mu.L sample of each of the standards, containing the calculated 
levels of FDP, was automatically added in the filling station of the 
instrument to 4.98 mL of 0.15M phosphate buffer, pH 7.8, containing 2.5% 
polyethylene glycol (molecular weight 6,000) and 0.025% sodium dodecyl 
sulfate. 60-.mu.L of rabbit anti-human fibrinogen antiserum (Cappel 
Laboratories) were added at breaker-mixer I, followed 3.5 minutes later by 
addition at breaker-mixer II of 50 .mu.L fibrinogen-PCMS particle reagent 
prepared in Example 2. The change in turbidity was measured at 340 nm, 39 
seconds and 56 seconds, respectively, after particle reagent addition, and 
the results presented in Table 5 as the extrapolated rate of change in 
milliabsorbance units over one minute. 
TABLE 5 
______________________________________ 
Fibrinogen Degradation Products 
Standard Curve 
Fibrinogen Degradation 
Rate 
Products (.mu.g/mL) 
(mAU/min at 340 nm) 
______________________________________ 
0 200 
10 115 
20 83 
40 65 
60 47 
100 30 
______________________________________ 
The data show that the turbidimetric rate achieved with fibrinogen-PCMS 
particle reagent and anti-fibrinogen antibody can be inhibited by adding 
increasing amounts of fibrinogen degradation products to produce a 
standard curve. Such a curve obtained with the particle reagent of this 
invention can be utilized to measure FDP in serum samples over the 
clinically useful range. 
Example 5 
Immobilization of Human Serum Albumin (HSA) on Particles Containing Various 
Levels of Chloromethylstyrene in the Shell 
(A) Preparation of Polystyrene/Polyvinylnaphthalene/Polychloromethylstyrene 
Core-Shell Particles 
An emulsion of polystyrene/polyvinylnaphthalene core polymer particles was 
prepared as in Example 1Aii. Fifty-two mL of the latex was used to prepare 
a core-shell latex in the same manner as in Example 1Aiii with 
chloromethylstyrene monomer. This core-shell polymer had substantially 
pure polychloromethylstyrene shell. 
(B) Preparation of 
Polystyrene/Polyvinylnaphthalene/Poly(chloromethylstyrene-Co-vinylnaphthal 
ene) Core-Shell Particles 
The procedure of Example 5A was repeated except for using a mixture of 10 
.mu.L of chloromethylstyrene and 90 mg of 2-vinylnaphthalene (VN) as shell 
monomers to provide a particle shell containing approximately 10% 
chloromethylstyrene and 90% VN. 
(C) Immobilization of HSA 
1 mL of latex from each of Examples 5A and 5B, in separate but identical 
procedures, was first mixed with 50 .mu.L of 10% sodium dodecyl sulfate 
solution and 4 mL of a 15 mM phosphate buffer, pH 7.5. This suspension was 
then added to a solution of 10 mg of HSA (Sigma Chemical Co.; 
crystallized, lyophilized, substantially globulin-free, an amount which is 
in excess of any available chloromethyl groups) in 15 mM phosphate buffer, 
pH 7.5. The pH of the mixture was adjusted to 7.8 using 0.1M sodium 
hydroxide, and incubated for the times and temperatures indicated in Table 
6. 
After the specified incubation time, each latex was centrifuged at 20,000 
RPM in a Sorvall.RTM. model RC-5B centrifuge. The supernatant was decanted 
from the pellet of particles, and the pellet resuspended in a 0.1% 
solution of GAFAC.RTM. RE610 in 15 mM phosphate buffer, pH 7.5, by 
sonication for 3 minutes with a Heat Systems Ultrasonics model 225R 
sonicator. The suspension was centrifuged a third time, and the pellet 
resuspended as above. The immobilized protein was measured by hydrolyzing 
1% solids latex samples for 20 minutes at 150.degree. C. with 6N 
hydrochloric acid and determining the amino acid content of the 
hydrolysate by the o-diphthalaldehyde method (see Methods in Enzymology, 
Vol. 91, p. 110). The results in Table 6 show that particle shell 
compositions containing either 10% or 100% chloromethylstyrene can 
covalently bind protein to approximately the same extent. Multiple washing 
steps with GAFAC.RTM. RE610 have been shown previously in Example 1 to 
remove adsorbed protein from particle reagents. To achieve similar protein 
levels in particle reagents at different incubation temperatures requires 
different incubation periods. 
TABLE 6 
______________________________________ 
Covalent Attachment of HSA to Latex Particles 
HSA 
Volume of Incubation 
Incubation 
Bound 
Type of 10% SDS Time Temperature 
to Latex 
Shell (.mu.L) (hour) (.degree.C.) 
(mg/mL) 
______________________________________ 
100% PCMS 
50 18 37 0.49 
50 168 4 0.48 
10/90% 50 18 37 0.45 
CMS/VN 50 168 4 0.42 
______________________________________ 
Example 6 
Preparation of Particle Reagent for Immunoassays for C-Reactive Protein 
(A) Polystyrene/Polyvinylnaphthalene Core Polymerization 
A latex of polyvinylnaphthalene was prepared in a 250 mL round-bottomed 
flask equipped with a magnetic stirrer and a reflux condenser. A 24-mL 
quantity of the polystyrene seed emulsion, prepared as in Example 1Ai, was 
added to 1.75 mL of water and then heated to 95.degree. C. This mixture 
was added to 20 g of 2-vinylnaphthalene (purified by sublimation and 
chromatography on basic alumina in a dichloromethane solution), 600 mg of 
sodium bicarbonate and 180 mg of potassium persulfate. As soon as the 
2-vinylnaphthalene had melted, 10 mL of 10% (w/v) sodium dodecyl sulfate 
solution was added to the mixture at a rate of 0.6 mL/min. One hour after 
the beginning of the SDS feed, the polymerization was complete. The 
optical density of the latex polymer measured at 340 nm was 1.128 after 
diluting 1:100 in water. The number average particle size was determined 
to be 39.4 nm by electron microscopy. The conversion of monomer to polymer 
was found to be 99.4% by gas chromatographic determination. 
(B) Shell Polymerizations 
Four different polymer particles were prepared containing different 
proportions of monomers in the shell as shown in Table 7. Each preparation 
began with 50 mL of latex from Example 6A and 1 g of a mixture of 
chloromethylstyrene and 2-vinylnaphthalene in the appropriate ratio to 
produce 10, 30, 70, and 100 percent (w/w) 
chloromethylstyrene/2-vinylnaphthalene shells (lots 6.1, 6.2, 6.3, and 
6.4, respectively, the latex from Example 6A having been designated as 
6.0). For each preparation, 50 mg of potassium persulfate was used in 
one-hour polymerizations at 95.degree.-100.degree. C. The monomer 
conversions and optical densities at 340 nm of a 1:100 dilution in water 
of the polymer particle latices are also shown in Table 7; the solids 
content was 14% 
TABLE 7 
______________________________________ 
Properties of Core-Shell Particles with Varying 
Ratio Chloromethylstyrene/Vinylnaphthalene Shells 
Chloro- 2-VN CMS Optical 
Pre- 2-Vinyl methyl- conver- 
conver- 
Density 
para- 
naphthalene 
styrene sion sion @ 340 nm 
tion (g) (mL) (%) (%) (1:100) 
______________________________________ 
6.0 0 0 -- -- 1.128 
6.1 0.90 0.10 99.93 &gt;97 1.734 
6.2 0.70 0.30 99.5 &gt;97 1.695 
6.3 0.30 0.70 99.90 &gt;97 1.640 
6.4 0 1.0 -- &gt;96 1.648 
______________________________________ 
(C) Preparation of Anti-C-Reactive Protein Antibody Particle Reagent 
Each of the polymer particle preparations 6.0-6.4 was used to prepare 
anti-C-reactive protein antibody particle reagents for use in direct 
immunoassays of CRP. 
Purified anti-CRP antibody (IgG) was prepared from immune rabbit serum by 
precipitation with ammonium sulfate adjusted to a final concentration of 
40% (w/v). The precipitate was collected by centrifugation (3000.times.g; 
20 minutes), dissolved in distilled water, precipitated and centrifuged a 
second time. The precipitate was dissolved in distilled water and dialyzed 
at 4.degree. C. overnight against 3 changes of 15 mM sodium phosphate 
buffer, pH 7.5. The level of the final IgG solution was calculated from 
280 nm absorbance and adjusted to 1 mg/mL. 
A 0.054-mL quantity of each polymer particle preparation was added to 2.05 
mL of the 1 mg/mL IgG solution in separate 10 mL Sorvall.RTM. centrifuge 
tubes. A 2.9-mL quantity of 15 mM phosphate buffer, pH 7.5, was added to 
each tube and the mixture incubated for 45 minutes at 37.degree. C. (Final 
IgG levels were 0.41 mg/mL in a buffer with 0.15% particle latex solids.) 
Preparation 6.0 (latex from Example 6A), without an outer shell, was 
treated in the same manner except that 0.069 mL of a 10.8% solids 
suspension was combined with 2.05 mL of 1 mg/mL antibody (IgG) solution in 
2.88 mL buffer. (The final IgG level was 0.41 mg/mL in a buffer with 0.19% 
particle latex solids.) 
The particles were centrifuged from suspension at 48,000.times.g for 90 
minutes at 4.degree. C. in a Sorvall.RTM. RC5B refrigerated centrifuge. 
The supernatant was decanted and the particle pellet resuspended in a wash 
solution of 50 mM glycine, pH 7.5, in volume equal to the supernatant. 
This wash step was repeated three times before a final resuspension in 200 
mM glycine, pH 7.5, was performed with one third the supernatant volume. 
This produced a 0.45% solids suspension of anti-CRP particle reagent. As 
expected, no particle reagent was obtained from preparation 6.0. 
(D) Immunoassay for CRP 
Each of the anti-CRP antibody particle reagent preparations from Example 6C 
was used to perform immunoassays for CRP in sodium phosphate buffer pH 
7.9, with 2% (w/v) PEG 8000. The assays were performed by mixing 25 .mu.L 
of anti-CRP particle reagent (from preparations 6.1-6.4 and the control, 
6.0) with 996 .mu.L of phosphate buffer having different molarities, 
ranging from 0.025 to 0.150M, as shown below in Table 8. After the 
solution had warmed to 37.degree. C., 4 .mu.L of normal human serum 
containing either 0 or 15 mg/dL CRP was added with mixing and the 340 nm 
absorbance of the reaction mixture was monitored with a Cary 219 (Varian 
Instruments) spectrophotometer for two minutes. 
The term separation is defined as the difference in absorbance between the 
0 and 15 mg/dL CRP level calibrators after a standard reaction period and 
is represented by the symbol .DELTA.. The data presented in Table 8 as 
milliabsorbance units show that separation for each shell composition is 
different at different phosphate buffer molarities. The maximum separation 
in the CRP assays performed was achieved with a 10% CMS/90% VN shell 
composition. 
TABLE 8 
__________________________________________________________________________ 
CRP Immunoassay Performance as a 
Function of Shell Composition 
Particle Reagent (% CMS in Shell) 
0 10 30 70 100 
Phos- 
CRP CRP CRP CRP CRP 
phate 
0 15 
.DELTA. 
0 15 .DELTA. 
0 15 .DELTA. 
0 15 .DELTA. 
0 15 .DELTA. 
(M) (mA) (mA) (mA) (mA) (mA) 
__________________________________________________________________________ 
0.025 
aggregated 
50 
840 
790 
5 290 
285 
0 100 
100 
20 150 
130 
0.050 
aggregated 
40 
720 
680 
8 220 
212 
-- 
ND -- 5 25 
20 
0.100 
0 20 
300 
280 
1 100 
99 
-- 
ND -- -- ND -- 
0.150 
0 10 
170 
160 
-- 
ND -- 0 25 
25 
0 15 
15 
__________________________________________________________________________