IMPROVED PROCEDURE FOR THE CONJUGATION OF RABBIT IGG AND FAB' ANTIBODIES WITH BETA-D-GALACTOSIDASE FROM ESCHERICHIA-COLI USING N,N'-ORTHO-PHENYLENEDIMALEIMIDE

Abstract

The procedures for the conjugation of rabbit Ig[immunoglobulin]G and Fab'' antibodies [Ab] with .beta.-D-galactosidase from E. coli using N,N''-o-phenylenedimaleimide were improved in several respects as compared with the previous methods. Maleimide residues were efficiently introduced into Ab under an atmosphere of N2; the average numbers of maleimide residues introduced into IgG and Fab'' Ab were 0.78 (0.65-0.86) and 0.86 (0.80-0.95)/molecule, respectively. The conjugation with the enzyme was performed at 4.degree. C at pH 6.5 for 15 or more h. The conjugates were almost completely separated from unreacted IgG and Fab'' by gel filtration. When the recoveries of IgG, Fab'' and .beta.-D-galactosidase in the conjugates were 23-29, 35-44 and 99%, respectively, the average numbers of IgG and Fab'' molecules conjugated with the enzyme were 1.5-1.7 and 2.1-2.8/molecule, respectively. There was no significant impairment of .beta.-D-galactosidase activity or the activity of anti-human IgG Ab to bind to human IgG upon conjugation. The conjugate preparation was heterogeneous and 1/3 of each preparation consisted of aggregated conjugates less useful in sandwich enzymoimmunoassay than the remaining material. The conjugate with Fab'' Ab gave lower control values in sandwich enzymoimmunoassay with silicone rubber as a solid phase than that with IgG Ab.