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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

使用合成肽作为抗原的抗 HTLV-I IgG 的新型超灵敏酶免疫分析（免疫复合物转移酶免疫分析）的实际应用研究

基本信息

批准号：
05557015
负责人：
ISHIKAWA Eiji
金额：
$ 4.54万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

以人工合成的多肽为抗原，建立了一种新的抗HTLV-Ⅰ IgG抗体的酶免疫分析方法（免疫复合物转移酶免疫分析法），并在灵敏度、特异性和性能方面进行了改进，使之能应用于实际检测。由2，4-二硝基苯-牛血清白蛋白-合成肽缀合物、抗HTLV-1抗体IgG和合成肽-β-D-半乳糖苷酶缀合物组成的免疫复合物被捕获到涂有亲和纯化的聚苯乙烯球上。（抗-2，4-二硝基苯基）IgG，用ε N-2洗脱，4-二硝基苯基-L-赖氨酸，并转移到涂有亲和纯化的聚苯乙烯的白色球上。通过荧光测定法测定与白色聚苯乙烯球结合的（抗人IgG γ链）IgG β-D-半乳糖苷酶活性。从检测HTLV-Ⅰ阳性血清IgG的9种合成肽中筛选出具有高灵敏度和高特异性的合成肽为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水平较低，低于Western印迹和免疫荧光测试可检测的水平。特异性可以通过与过量的合成肽预孵育来证实，所述合成肽在不存在去污剂的情况下是可溶的，并且通过证明不仅在血清中而且在尿液中以及可能在来自HTLV-I的两种或更多种不同肽的口腔液中的HTLV-I的抗体IgG来改善。该方法可用于检测HTLV-I阳性血清的IgG抗体。为了实际应用，通过使用微孔板和荧光读数器简化了该测定方法的过程，消除了使用镊子将固相从试管转移到试管，这是由于残留导致假阳性的原因。人们仍在尝试改变固相的形式以用于更实际的应用。