Enzyme‐Linked Sandwich Immunoassay of Macromolecular Antigens Using the Rabbit Antibody‐Coupled Glass Rod as a Solid Phase

Abstract

A highly sensitive sandwich immunoassay of macromolecular antigens using the rabbit antibody Fab′—β‐d‐galactosidase complex and the rabbit antibody immunoglobulin‐G‐coupled glass rod as a solid phase is described. The Fab′ fragments of rabbit antibody IgG are conjugated with β‐d‐galactosidase from Escherichia coli using N,N′‐o‐phenylenedimaleimide. Rabbit antibody IgG is coupled to the aminoalkylsilyl glass rods (3 mm in diameter and 5 mm in length) using glutaraldehyde. A wide range of the concentrations of rabbit IgG fraction (20–2000 μg/ml) is effective for coupling, and the amount of rabbit immunoglobulin G coupled can be controlled. The smallest amounts of ornithine σ‐aminotransferase from rat liver, human immunoglobulin G and 2,4‐dinitrophenyl human immunoglobulin G that can be determined are 0.03, 0.3 and 0.04 fmol, respectively. The sensitivity of the assay for these antigens is affected mainly by the non‐specific binding of the complexes to the solid phase and the ability of antigen molecules, adsorbed on the solid phase, to bind specifically the complexes. The assay with the rabbit antibody immunoglobulin‐G‐coupled glass rods is simpler and more sensitive than that with the rabbit antibody immunoglobulin‐G‐coupled Sepharose 4B.