Sensitive detection of anti-human T-cell leukemia virus type I IgG in human serum by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag-env hybrid protein as antigen

Abstract

Anti-human T-cell leukemia virus type I IgG (anti-HTLV-I IgG) in human serum was detected with high sensitivity by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag(114–139)- env (197–295) hybrid protein. Anti-HTLV-I IgG in test serum was reacted simultaneously with dinitrophenyl bovine serum albumin-recombinant gag-env hybrid protein conjugate and recombinant gag-env hybrid protein-horseradish peroxidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified anti-dinitrophenyl group IgG. After washing the polystyrene balls to eliminate nonspecific IgG in the test serum and excess of the peroxidase conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified anti-human IgG 7-chain IgG. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The nonspecific binding of peroxidase activity was remarkably reduced by transfer of the complex and the detection limit of anti-HTLV-I IgG in serum was lowered 300 to 3000-fold compared with that by Western blotting and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with the test serum and, after washing, with anti-human IgG γ-chain Fab'-peroxidase conjugate. The immune complex transfer enzyme immunoassay may overcome some difficulties with currently used methods.