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

本文建立了一种以合成肽为抗原检测HTLV-I抗体IgG的新型酶免疫分析法（免疫复合物转移酶免疫分析法），并在灵敏度、特异性和性能上进行了改进，可用于实际检测。由2,4-二硝基苯-牛血清白蛋白-合成肽偶联物、htlc - i抗体IgG和合成肽- β -d -半乳糖苷酶偶联物组成的免疫复合物被捕获在涂有亲和纯化（抗2,4-二硝基苯基）IgG的有色聚苯乙烯球上，用epsilonn -2,4-二硝基苯-l -赖氨酸洗脱，转移到涂有亲和纯化（抗人IgG γ链）IgG的白色聚苯乙烯球上。用荧光法测定与白色聚苯乙烯球结合的β - d -半乳糖苷酶活性。从HTLV-I血清阳性血清样品中检测HTLV-I抗体IgG的9条多肽中选择的高灵敏度和高特异性合成多肽分别为Ac-Cys-env gp46（188-224）、Ala-Cys-env gp46（237-262）和Cys-gag p19（100-130）。该方法检测到的HTLV-I抗体IgG水平低于Western blotting和免疫荧光法。这种特异性可以通过合成多肽的预孵育来证实，这些合成多肽在没有洗涤剂的情况下是可溶的，并且通过在血清、尿液和可能在口液中发现HTLV-I对两种或两种以上不同多肽的抗体IgG来提高。该方法可检测出HTLV-I血清阳性抗体IgG。在实际应用中，该分析方法通过使用微孔板和荧光阅读器简化了过程，消除了使用镊子将固相从试管转移到试管的情况，这是由于携带而导致假阳性的原因。为了更实际的应用，人们仍在尝试改变固相的形态。

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.