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

循环中许多多肽的浓度仅在提取和浓缩后通过竞争性放射免疫分析法确定。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多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（异种位点酶免疫分析方法）。方法1将>肽生物素化，并将其捕获在抗肽IgG包被的聚苯乙烯球上。将聚苯乙烯球洗涤去除其他生物素化物质后，用盐酸将生物素化肽从聚苯乙烯球中洗脱，与抗肽Fab′-过氧化物酶偶联物反应。形成的络合物被捕获在链亲和素涂层的聚苯乙烯球上。用荧光法测定与聚苯乙烯球结合的过氧化物酶活性。方法二从上述抗肽IgG包被的聚苯乙烯球中洗脱>Bjotinylated肽，再与2,4 -二硝基苯-荧光素二硫-牛血清白蛋白-兔抗肽IgG偶联物反应。形成的配合物被捕获在IgG包被的聚苯乙烯球上，洗涤后与亲和素- β - D -半乳糖苷酶偶联物反应。对聚苯乙烯球进行洗涤，用epsilon N - 2,4 -二硝基苯- L -赖氨酸洗脱三组分的配合物，转移到抗荧光素IgG包被的聚苯乙烯球上。洗涤后，配合物通过2 -巯基乙胺还原从聚苯乙烯球中释放出来，转移到IgG包被的聚苯乙烯球上。最后一个聚苯乙烯球结合的β - D -半乳糖苷酶活性用荧光法测定。[结果]方法1和方法11对[Arg^8] -抗利尿素、α -房利钠肽和血管紧张素1的检出限分别为10amol和1amol。这两种方法的原理可适用于测量包括可衍生以同时被抗半抗原抗体和亲和素（链亲和素）分子结合的肽的速食剂。少

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