Novel and sensitive enzyme immunoassay (immune‐complex‐transfer enzyme immunoassay) for antihuman t cell leukemia virus type 1 igg in human serum using recombinant gag‐env hybrid protein as antigen

Abstract

A novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG (anti-HTLV-1 IgG) in human serum using recombinant gag(14-139)-env-(197-295) hybrid protein is described. Anti-HTLV-1 IgG in test serum was reacted with dinitrophenyl biotinyl bovine serum albumin-recombinant gag-env hybrid protein conjugate. The complex formed was trapped onto polystyrene balls coated with affinity-purified antidinitrophenyl group IgG. After washing to eliminate nonspecific IgG in the test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with streptavidin. After washing, anti-HTLV-1 IgG in the complex trapped onto the streptavidin-coated polystyrene balls was reacted with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. By transfer of the complex, the nonspecific binding of nonspecific human IgG was considerably reduced, and the detection limit of anti-HTLV-1 IgG in serum was lowered 30-300-fold compared with that by Western blotting, gelatin particle agglutination, and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with test serum and, after washing, with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Usefulness of the immune-complex-transfer enzyme immunoassay was demonstrated using 271 serum samples.