Nephelometric determination of HB.sub.s antigen with antibody-containing microparticles

A reagent kit for nephelometrically determining antigen by contacting antigen with antibody or antibody fragments, which comprises antibody or antibody fragment containing polyfunctional hydrophilic spherical microparticles of uniform size having an average diameter of between about 10 nanometers and about 10 micrometers, said microparticles comprise copolymers obtained by copolymerizing:

• (a) from 40 to 60% by weight of the total monomers of an acrylic aldehyde having a formula ##STR18## in which R.sub.1 is an alkyl radical having from 1 to 4 carbon atoms, (b) from 40 to 70% by weight to total monomers of an acrylic acid derivative having a formula ##STR19## in which R.sub.2 is hydrogen or an alkyl group having from 1 to 4 carbon atoms and R.sub.3 is a lower hydroxyalkyl radical,
• (c) and less than 15% by weight of total monomers of at least one acrylic acid derivative having a formula:
• ##STR20## wherein R.sub.2 is hydrogen or lower alkyl and R.sub.4, R.sub.5 and R.sub.6, which may be the same or different, represent a C.sub.1-4 alkyl group;
• and from 0.1 to 10% by weight of total monomers of an unconjugated diene crosslinking agent;
• said microparticles having coupled thereto an antibody or a fragment of an antibody having the ability to recognize the specific antigen and selected from the group consisting of polyclonal antibodies isolated from specific immune sera obtained from animals after immunization with purified HB.sub.s antigen and monoclonal antibodies produced from hybrid cells resulting from the fusion of plasmocytes of animals immunized against HB.sub.s antigen, and from myeloma cells.

A method for making antibody containing polyfunctional hydrophilic spherical microparticles of uniform size having an average diameter of between about 10 nanometers and about 10 micrometers, which comprises copolymerizing:

• (a) from 5 to 95% by weight of the total monomers of an acrylic aldehyde having a formula ##STR21## in which R.sub.1 is an alkyl radical having from 1 to 4 carbon atoms, (b) from 5 to 90% by weight of total monomers of an acrylic acid derivative having a formula ##STR22## in which R.sub.2 is hydrogen or an alkyl group having from 1 to 4 carbon atoms and R.sub.3 is a lower hydroxyalkyl radical,
• (c) and less than 15% by weight of total monomers of at least one acrylic acid derivative having a formula:
• ##STR23## wherein R.sub.2 is hydrogen or lower alkyl and R.sub.4, R.sub.5 and R.sub.6, which may be the same or different, represent a C.sub.1-4 alkyl group;
• and from 0.1 to 10% by weight of total monomers of an unconjugated diene crosslinking agent to form said microparticles and then coupling thereto an antibody selected from the group consisting of polyclonal antibodies isolated from specific immune sera obtained from animals after immunization with purified antigen and monoclonal antibodies produced from hybrid cells resulting from the fusion of plasmocytes of animals immunized against antigen, and from myeloma cells to obtain said antibody containing microparticles.