Water-insoluble particle and immunoreactive reagent, analytical elements and methods of use

Claim 2

A water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and which has a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell comprising a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): X is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent. ##STR5## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR6## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- of --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.

Claim 3

The particle of claim 1 having a diameter of from about 0.05 to about 5 micrometers.

Claim 4

The particle of claim 1 wherein said monomers from which said --F-- units of said second polymer are derived have reactive groups selected from the group consisting of halomethyl, activated 2-substituted ethylsulfonyl and vinylsulfonyl reactive groups.

Claim 5

The particle of claim 1 wherein said monomers represented by D include at least one ethylenically unsaturated polymerizable monomer having anionic groups.

Claim 6

The particle of claim 1 wherein said inner core is from about 30 to about 80 percent based on the total polymer particle weight.

Claim 7

The particle of claim 1 wherein said detectable tracer is selected from the group consisting of a colorimetric compound, fluorometric compound and a dye-forming compound.

Claim 8

The polymeric particle of claim 1 wherein said outer shell is swellable in water-miscible solvents selected from the group consisting of acetonitrile, N,N-dimethylformamide, lower alcohols, tetrahydrofuran, ketones and 1-methyl-2-pyrrolidinone.

Claim 9

An immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell composed of a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ), greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): --(A).sub.u --(B).sub.v --(D).sub.w -- wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): --(E).sub.x --(F).sub.y --(G).sub.z -- wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species; --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.

Claim 10

An immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell composed of a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ), greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): --(A).sub.u --(B).sub.v --(D).sub.w -- wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): --(E).sub.x --(F).sub.y --(G).sub.z -- wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species; --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.

Claim 11

The reagent of claim 8 wherein said immunologically reactive species is a drug, hormone, antibiotic, antibody or other compound having antigenic properties.

Claim 12

The reagent of claim 8 wherein said immunologically reactive species is an antibody.

Claim 13

The reagent of claim 8 wherein said monomers from which --F-- recurring units are derived have reactive groups selected from the group consisting of halomethyl, activated 2-substituted ethylsulfonyl and vinylsulfonyl reactive groups.

Claim 14

The reagent of claim 8 wherein said detectable tracer is selected from the group consisting of a colorimetric compound, fluorometric compound and a dye forming compound.

Claim 15

An analytical element comprising an absorbent carrier material having one or more zones, and containing in one or more of said zones an immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell comprising a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR7## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR8## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.

Claim 16

An analytical element comprising an absorbent carrier material having one or more zones, and containing in one or more of said zones an immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell comprising a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR7## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR8## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.

Claim 17

The element of claim 13 wherein said immunoreactive species is an antibody.

Claim 20

The method of claim 15 wherein said reaction product is separated from said unreacted materials prior to said determination step.

Claim 21

The method of claim 15 for the determination of Streptococcus A antigen in a biological sample wherein said immunoreactive species is an antibody to Streptococcus A antigen.

Claim 22

The method of claim 15 for the determination of hCG in a biological sample wherein said immunoreactive species is an antibody to hCG.

Claim 23

The method of claim 15 for the determination of antibodies against a compound of biological interest selected from the group consisting of HTLV and HIV in a biological sample wherein said immunoreactive species is selected from the group consisting of a HTLV and a HIV antigen, respectively.

Claim 24

The method of claim 15 wherein, prior to, simultaneously with or subsequent to said contacting step A, contacting said biological compound with a second immunoreactive species which is capable of participating in a specific binding reaction with said compound but which is not reactive with said immunoreactive species of said reagent.

Claim 25

An agglutination method for the determination of Streptococcus A antigen in an aqueous liquid comprising: A. contacting an extraction solution of Streptococcus A organisms with an immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell composed of a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups selected from the group consisting of active halogen atoms, carboxyl, epoxy, isocyanate, amine, aziridine, 2-substituted ethylcarbonyl, aldehyde, activated 2-substituted ethylsulfonyl and vinylsulfonyl, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR9## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR10## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an antibody to Streptococcus A antigen, product of said antigen and said antibody, B. determining the amount of tracer either in said agglutinate or in unagglutinated materials.

Claim 26

An agglutination method for the determination of Streptococcus A antigen in an aqueous liquid comprising: A. contacting an extraction solution of Streptococcus A organisms with an immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell composed of a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups selected from the group consisting of active halogen atoms, carboxyl, epoxy, isocyanate, amine, aziridine, 2-substituted ethylcarbonyl, aldehyde, activated 2-substituted ethylsulfonyl and vinylsulfonyl, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR9## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR10## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an antibody to Streptococcus A antigen, product of said antigen and said antibody, B. determining the amount of tracer either in said agglutinate or in unagglutinated materials.

Claim 27

An aqueous suspension of a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell comprising a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR11## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR12## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent.

Claim 28

An aqueous suspension of a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell comprising a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR11## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 mole percent, and said second polymer is represented by the formula (II): ##STR12## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent.

Claim 29

An aqueous suspension of an immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell composed of a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR13## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55 ° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 percent, and said second polymer is represented by the formula (II): ##STR14## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, tracer has a high affinity and which has a x is from 45 to 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.

Claim 30

An aqueous suspension of an immunoreactive reagent comprising: (a) a water-insoluble polymeric particle having: an inner core comprising a detectable tracer distributed within a first polymer for which said tracer has a high affinity and having a glass transition temperature (Tg 1 ) of less than about 100° C., said first polymer being derived from one or more ethylenically unsaturated polymerizable monomers, and an outer shell composed of a second polymer for which said tracer has substantially less affinity relative to said first polymer, said second polymer having a glass transition temperature (Tg 2 ) greater than or equal to the term (Tg 1 -10° C.), and being derived from one or more ethylenically unsaturated polymerizable monomers, at least one of which monomers has reactive groups which are directly or indirectly reactive with free amino or sulfhydryl groups of an immunoreactive species, provided that said outer shell is swellable in water-miscible organic solvents, wherein said first polymer is represented by the formula (I): ##STR13## wherein --A-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated monomers which provide water insolubility to said particles, --B-- represents recurring units derived from one or more ethylenically unsaturated monomers whose homopolymers have a glass transition temperature less than about 55 ° C., --D-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --A-- or --B--, u is from about 30 to about 95 mole percent, v is from 5 to about 50 mole percent, and w is from 0 to 20 percent, and said second polymer is represented by the formula (II): ##STR14## wherein --E-- represents recurring units derived from one or more hydrophobic ethylenically unsaturated aromatic monomers, --F-- represents recurring units derived from one or more ethylenically unsaturated monomers having reactive groups which will directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, tracer has a high affinity and which has a x is from 45 to 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent directly or indirectly react with said free amine or sulfhydryl groups of said immunoreactive species, --G-- represents recurring units derived from one or more ethylenically unsaturated monomers which are different than those represented by --E-- or --F--, x is from 45 to about 99 mole percent, y is from about 1 to 50 mole percent, and z is from 0 to about 5 mole percent, said particle having substantially none of said tracer within said outer shell or on its outer surface, and said particle being covalently attached through said reactive groups on the outer surface to (b) an immunoreactive species which is capable of participating in an immunological reaction with a compound of biological interest.