Use of Rabbit Antibody IgG-loaded Silicone Pieces for the Sandwich Enzymoimmunoassay of Macromolecular Antigens

A highly sensitive sandwich enzymoimmunoassay of macromolecular antigens using the Fab' of rabbit antibody coupled to -β-D-galactosidase together with rabbit antibody IgG loaded on silicone pieces is described. The Fab' fragments of rabbit antibody IgG are conjugated with β-D-galactosidase from Escherichga coli using N,N'-o-phenylenedimaleimide. Small silicone pieces are loaded with rabbit antibody IgG by simple adsorption. A wide range of concentrations of rabbit antibody IgG fraction (2 to 2000 μg/ml) is effective for loading. The adsorp tion is sufficiently stable for immunoassay and the amount of rabbit antibody IgG loaded can be controlled. The smallest amounts of ornithine δ-aminotransferase from rat liver, human IgG, and DNP-human IgG that could be determined were 0.03, 0.3, and 0.03 fmol, respectively The sensitivity of the assay for these antigens is affected mainly by the non-specific binding of complexes to the solid phase and by the ability of antigen molecules, when adsorbed on the solid phase, to bind with the complexes specifically. The assay with rabbit antibody IgG loaded on silicone pieces is simpler and more sensitive than that with rabbit antibody lgG coupled to Sepharose 4B and is as sensitive as that with rabbit antibody IgG coupled to glass rods. Suita ble silicone pieces can be more readily prepared in the laboratory than glass rods. The F(ab')2, not Fab, fragments may also be used to load silicone pieces in a similar manner for immuno assay.