Biologically active reagent prepared from aldehyde-containing polymer, test kit, analytical element and methods of use

An analytical element comprising a nonporous support, having disposed thereon, in order and in fluid contact,

• a reagent layer containing one or more reagents for providing a detectable signal in response to a ligand of interest, and
• a porous spreading layer,
• said element further containing, in one or more layers, a biologically active reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a homopolymer or copolymer derived by emulsion or suspension polymerization from an ethylenically unsaturated poolymerizable monomer having a pendant aldehyde group and represented by the structure:
• ##STR5## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) a receptor for said ligand covalently attached to said particle through said pendant aldehyde group.

A method for the determination of a specific binding ligand comprising:

• A. forming a water-insoluble specific binding complex of a specific binding ligand of interest, or a receptor therefor, with a reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a homopolymer or copolymer derived by emulsion or suspension polymerization from an ethylenically unsaturated polymerizable monomer having a pendant aldehyde group and represent by the structure:
• ##STR6## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) a biologically active substance covalently attached to said particle through said pendant aldehyde group, said biologically active substance being specifically reactive with either said ligand or a receptor therefor, and
• B. detecting the presence of said complex as an indication of the presence of amount of said ligand.

A competitive binding assay for the determination of a water-soluble specific binding ligand comprising:

• A. contacting a specimen suspected of containing a water-soluble specific binding ligand of interest with both
• a water-soluble specific receptor for said ligand, and
• a reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a homopolymer or copolymer derived by emulsion or suspension polymerization from an ethylenically unsaturated polymerizable monomer having a pendant aldehyde group and represented by the structure:
• ##STR7## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) molecules of said ligand covalently attached to said particle through said pendant aldehyde group,
• to form a water-soluble specific binding complex (a) between said receptor and said ligand, and a water-insoluble specific binding complex (b) between said receptor and said water-soluble reagent, and
• B. after separating complexes (a) and (b), detecting the presence of either complex as a determination of said ligand.

A competitive binding assay for the determination of a specific binding ligand comprising:

• A. contacting a specimen suspected of containing a water-soluble specific binding ligand of interest with both
• a water-soluble detectable analog of said ligand, and
• a reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a homopolymer or copolymer derived by emulsion or suspension polymerization from an ethylenically unsaturated polymerizable monomer having a pendant aldehyde group and represented by the structure:
• ##STR8## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) a receptor for said ligand covalently attached to said particle through said pendant aldehyde group,
• to form a water-insoluble specific binding complex (a) between said water-insoluble receptor and said ligand, and water-insoluble specific binding complex (b) between said water-insoluble receptor and said ligand analog, and
• B. after separating complexes (a) and (b), detecting the presence of either complex as a determination of said ligand.

A method for the determination of an immunological species comprising:

• A. contacting a specimen suspected of containing an immunological species with a reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a homopolymer or copolymer polymer derived by emulsion or suspension polymerization from an ethylenically unsaturated polymerizable monomer having a pendant aldehyde group and represented by the structure:
• ##STR9## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) a receptor for said species covalently attached to said particle through said pendant aldehyde group,
• to form a water-insoluble immunological complex of said species with said receptor, and
• B. after separating uncomplexed materials from said water-insoluble complex, detecting the presence of said water-insoluble complex as an indication of the presence or amount of said immunological species in said specimen.

An analytical separation method comprising:

• A. passing a specimen containing a mixture of biologically active substances over an affinity chromatography reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a copolymer or homopolymer derived by emulsion or suspension polymerization from an ethylenically unsaturated polymerizable monomer having a pendant aldehyde group and represented by the structure:
• ##STR10## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) a specific binding substance covalently attached to said particle through said pendant aldehyde group, said specific binding substance being specific to one or more predetermined biologically active substances in said specimen mixture of biologically active substances
• to form a complex of said reagent with said predetermined biologically active substances, and
• B. collecting either the one or more complexed predetermined substances or one or more substances remaining in the eluent.

A method for the detection of a nucleic acid comprising:

• A. forming a water-insoluble hybridization product between a nucleic acid of interest and a reagent comprising:
• (I) a water-insoluble particle which is non-swellable in water, and consists essentially of a homopolymer or copolymer polymer derived by emulsion or suspension polymerization from an ethylenically unsaturated polymerizable monomer having a pendant aldehyde group and represented by the structure:
• ##STR11## wherein Ar is arylene, R and R.sup.1 are independently hydrogen, halo or lower alkyl, R.sup.2 and R.sup.3 are independently alkylene of 1 to 4 carbon atoms in the chain, R.sup.4 is arylene, m is 0 or 1, and n is an integer of 1 to 4, and
• (II) an oligonucleotide covalently attached to said particle through said pendent aldehyde group, said oligonucleotide being complementary to said nucleic acid of interest, and
• B. detecting the hybridization product as a determination of said nucleic acid of interest.