Studies for the Practical Use of Novel and Ultrasensitive Enzyme Immunoassay (Immune Complex Transfer Enzyme Immunoassary) of Anti-HTLV-I IgG Using Synthetic Peptides as Antigens

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A novel enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibody IgG to HTLV-I using synthetic peptides as antigens has been developed and improved in sensitivity, specificity and performance so as to be used in practical assays. The immune complex, consisting of 2,4-dinitrophenyl-bovine serum albumin-synthetic peptide conjugate, antibody IgG to HTLV-I and synthetic peptide-beta-D-galactosidase conjugate was trapped onto colored polystirene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG,was eluted with epsilonN-2,4-dinitrophenyl-L-lysine and was transferred onto white polystirene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG.beta-D-Galactosidase activity bound to the white polystirene balls was assayd by fluorometry. The syntehtic peptides selected from nine peptides tested for detecting antibody IgG to HTLV-I in serum samples of HTLV-I seropositives with high sensitivity and high specificity were Ac-Cys-env gp46 (188-224) , Ala-Cys-env gp46 (237-262) and Cys-gag p19 (100-130) . This method detected antibody IgG to HTLV-I at low levels below those detectable by Western blotting and immunofluorescence test. The specificity could be confirmed by preincubation with excess of the synthetic peptides, which were soluble in the absence of detergents, and was improved by demonstrating antibody IgG to HTLV-I not only in serum but also in urine and probably in oral fluids from HTLV-I to two or more different peptides. This method could detect antibody IgG to HTLV-I seropositives. For practical use, this assay method was simplified in processes by using microplates and a fluororeader, eliminating the use of tweezers for transfer of solid phase from test tube to test tube, which was causative of false-positivity due to carryover. Attempts are still being made to modify the form of solid phase for more practical use.