Detection of one attomole of [Arg8]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay).
Detection of one attomole of [Arg8]-vasopressin by novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay).
复制标题
通过新型非竞争性酶免疫分析（异二位点复合转移酶免疫分析）检测一阿托摩尔的 [Arg8]-加压素。
DOI：
10.1093/oxfordjournals.jbchem.a123608
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发表时间：
1991
影响因子：
2.7
通讯作者：
E. Ishikawa
中科院分区：
文献类型：
--
作者：
S. Hashida;K. Tanaka;N. Yamamoto;T. Uno;K. Yamaguchi;E. Ishikawa
用一种新的非竞争性酶免疫分析法（异源双位点复合物转移酶免疫分析法）检测1阿摩尔[Arg 8]-加压素（AVP）。使用N-羟基琥珀酰亚胺生物素间接生物素化AVP，并将其捕获到抗AVP IgG包被的聚苯乙烯球上。洗涤后，用HCl从聚苯乙烯球上洗脱生物素化的AVP，并与2，4-二硝基苯基-荧光素二硫化物-牛血清白蛋白-兔抗AVP IgG缀合物反应。将形成的复合物捕获在[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-巯基乙胺还原将复合物从聚苯乙烯球中释放出来，并转移到[抗兔IgG] IgG包被的聚苯乙烯球中。通过荧光测定法测定与最后的聚苯乙烯球结合的β-D-半乳糖苷酶活性。AVP的检测限为1.1 fg（1阿莫尔）/管。通过使用分子筛从蛋白质中分离肽来消除生物流体中蛋白质的干扰。本方法的原理可适用于测量半抗原，包括肽，其可被衍生化以同时被抗半抗原抗体和抗生物素蛋白分子结合。
One attomole of [Arg8]-vasopressin (AVP) was detected by a novel noncompetitive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). AVP was indirectly biotinylated using N-hydroxysuccinimidobiotin and trapped onto an anti-AVP IgG-coated polystyrene ball. After washing, biotinylated AVP was eluted from the polystyrene ball with HCl and was reacted with 2,4-dinitrophenyl-fluorescein disulfide-bovine serum albumin-rabbit anti-AVP IgG conjugate. The complex formed was trapped on [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 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. The detection limit of AVP was 1.1 fg (1 amol)/tube. Interference by proteins in biological fluids was eliminated by separation of peptides from proteins using a molecular sieve. The principle of the present method may be applicable to the measurement of haptens, including peptides, that can be derivatized so as to be bound simultaneously by both anti-hapten antibody and avidin molecules.