Conjugation of recombinant reverse transcriptase of HIV-1 to β- d-galactosidase from Escherichia coli for ultrasensitivi enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HIV-1 IgG

Abstract

Recombinant reverse transcriptase (RT) of HIV-1 was conjugated to β- d-galactosidase from Escherichia coli in three different ways. Maleimide groups were introduced into β- d-galactosidase molecules using N, N′- o-phenylenedimaleimide in the absence (method I) or presence (method II) of N-ethylmaleimide or into β- d-galactosidase molecules, which had been treated with excess of 4,4′-dithiopyridine to block thiol groups, using N-succinimidyl-6-maleimidohexanoate (method III). Subsequently, the maleimide groups were reacted with thiol groups introduced into recombinant RT molecules using N-succinimidyl- S-acetylmercaptoacetate. The conjugates were tested by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay). The immune complex consisting of 2,4-dinitrophenyl-bovine serum albumin-recombinant RT conjugate, anti-HIV-1 IgG and recombinant RT-β- d-galactosidase conjugate was captured by polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG, eluted with N ϵ-2,4- dinitrophenyl- l-lysine and transferred to polystyrene beads coated with (anti-human IgG γ chain) IgG. The conjugate prepared by method III, which showed the least polymerization, the least loss of the specific enzyme activity and the lowest nonspecific binding, improved the sensitivity of the enzyme immunoassay for anti-HIV-1 IgG approximately 30-fold compared with RT-horseradish peroxidase conjugate.