HYDROPHILIZED AND FUNCTIONALIZED MICROTITER PLATES FOR THE SITE-SPECIFIC COUPLING OF ANTIGENS AND ANTIBODIES - APPLICATION TO THE DIAGNOSIS OF VIRAL CARDIAC AND AUTOIMMUNE-DISEASES

Extensive adsorption of macromolecules (antigens and antibodies) took place when they were covalently bound to non-hydrophilized polystyrene microtiter plates. Precoating polystyrene surfaces with gelatin reduced this non-specific adsorption only partially, whereas precoating with uncharged polymers such as poly(ethylene glycol) (PEG) and polysaccharides eliminated the problem totally. On such hydrophilized plates functionalized with epoxide groups, antigens and antibodies were randomly bound. On those functionalized with acid hydrazide, antibodies were site-specifically bound by their carbohydrate residues. These site-specifically bound antibodies retained greater reactivity and more of their native antigenic structure than randomly coupled antibodies. They therefore permitted the measurement of a minor analyte (D-dimer) in the presence of an excess of major components such as fibrinogen. Enzyme immunoassays which were uninterpretable on non-hydrophilized plates because of the adsorption of immunoglobulin gave meaningful results on hydrophilized plates. This held true for immunoglobulin aggregates formed artificially either by successive cycles of freezing and thawing or by the acid treatment of serum to dissociate immune complexes. The latter approach permitted us to obtain unequivocal evidence for the presence of antibodies specific for human T-cell lymphotropic virus type 1 (HTLV1) in immune complexes in sera with no detectable free antibody. For naturally occurring aggregates which are often found in the sera of patients with autoimmune diseases, the use of hydrophilized plates also permitted antibody levels to be measured in instances where background noise was greater than the signal on non-hydrophilized plates. This combination of hydrophilization and site-specific coupling, of monoclonal antibodies of a defined specificity should provide a distinct advantage when developing routine covalent enzyme linked immunoassays (CELIAs) for minor analytes, in a mixture with major constituents, as is often encountered, in both human and veterinary medicine and in the food industry.