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Basic Study on the Development and Application of Novel and Ultrasensitive Immunoassay for Haptens, Especially, Small Peptide Molecules

半抗原特别是小肽分子新型超灵敏免疫分析技术的开发和应用基础研究

基本信息

批准号：
02808029
负责人：
HASHIDA Seiichi
金额：
$ 0.96万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The concentration of many peptides in the circulation has been determined by competitive radioimmunoassay only after extraction and concentration. This is due to insufficient sensitivities of competitive immunoassays. The purpose of this research was to develop noncompetitive immunoassay methods for peptides. [Methods]Two noncompetitive enzyme immunoassay methods (hetero - two - site enzyme immunoassy methods) were developed. <Method I>Peptide was biotinylated and trapped onto an anti - peptide IgG - coated polystyrene ball. After washing the polystyrene ball to eliminate other biotinylated substances, biotinylated peptide was eluted from the polystyrene ball with HCl and reacted with anti -peptide Fab' -peroxidase conjugate. The complex formed was trapped onto streptavidin - coated polystyrefie balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. <Method II>Bjotinylated peptide, eluted from anti - peptide IgG - coated polystyrene ball as described abov … More e, was reacted with 2, 4 - dinitrophenyl - fluorescein disulfide - bovine serum albumin - rabbit antipeptide IgG conjugate. The complex formed was trapped onto[anti - 2, 4 - dinitrophenyl group]IgG - coated polystyrene balls and, after washing, reacted with avidin - beta - D -galactosidase conjugate. The polystyrene balls were washed, and the complex of the three components was eluted with epsilon N - 2, 4 - dinitrophenyl - L - lysine and transferred to anti - fluorescein IgG - coated polystyrene balls. After washing, the complex was released from the polystyrene balls by reduction with 2 - mercaptoethylamine and transferred to[anti - rabbit IgG]IgG - coated polystyrene balls. beta - D - Galactosidase activity bound to the last polystyrene balls was assayed by fluorometry. [Results]The detection limits by Methods I and 11 for peptides such as[Arg^8] - vasopressin, alpha - atrial natriuretic peptide and angiotensin I were 10 amol and I amol, respectively. The principle of the two methods may be applicable to the measurement of hastens including peptides that can be derivatized so as to be bound simultaneously by both anti - hapten antibody and avidin(streptavidin)molecules. Less

许多肽在循环中的浓度仅在提取和浓缩后才通过竞争性放射免疫测定法测定。这是由于竞争性免疫测定的灵敏度不足。本研究的目的是开发肽的非竞争性免疫测定方法。[方法]建立了两种非竞争性酶免疫分析方法（异源双位点酶免疫分析法）。<Method I>将肽生物素化并捕获到抗肽IgG包被的聚苯乙烯球上。洗涤聚苯乙烯球以除去其它生物素化物质后，用HCl从聚苯乙烯球上洗脱生物素化肽，并与抗肽Fab ′-过氧化物酶缀合物反应.将形成的复合物捕获到链霉亲和素包被的聚苯乙烯球上。过氧化物酶活性结合到聚苯乙烯球测定荧光法。<Method II>从如上所述的抗肽IgG包被的聚苯乙烯球上洗脱的Bjotinylated肽 ...更多信息用2，4 -二硝基苯基-二硫代荧光素-牛血清白蛋白-兔抗肽IgG偶联物进行反应。将形成的复合物捕获到[抗-2，4-二硝基苯基]IgG包被的聚苯乙烯球上，洗涤后，与抗生物素蛋白-β- D -半乳糖苷酶缀合物反应.洗涤聚苯乙烯球，用1 N - 2，4 -二硝基苯基- L -赖氨酸洗脱三种组分的复合物，并转移到抗荧光素IgG包被的聚苯乙烯球上.洗涤后，通过用2 -巯基乙胺还原从聚苯乙烯球释放复合物，并转移到[抗兔IgG]IgG包被的聚苯乙烯球上。通过荧光测定法测定与最后一个聚苯乙烯球结合的β- D -半乳糖苷酶活性。[结果]方法I和方法11对[Arg ^8] -加压素、α-心钠素和血管紧张素I等肽类的检测限分别为10阿莫尔和1阿莫尔。这两种方法的原理可适用于测定促生物素，所述促生物素包括可被衍生化以同时被抗半抗原抗体和抗生物素蛋白（链霉抗生物素蛋白）分子结合的肽。少