Yukawa N, Kohno T, Osawa M, Saito T, Nakajima Y, Ishikawa E, Takahama K, Takeichi S
Department of Forensic Medicine, Tokai University School of Medicine, Kanagawa, Japan.
J Immunoassay. 1995 Aug;16(3):247-61. doi: 10.1080/15321819508013561.
Sandwich capture enzyme immunoassays (EIA I and EIA II) are described. Water soluble B substance was reacted simultaneously with affinity-purified dinitrophenyl goat anti-B IgG and affinity-purified goat anti-B IgG-peroxidase conjugate. The complex formed of B substance, dinitrophenyl IgG and IgG-peroxidase conjugate was trapped onto a polystyrene ball coated with affinity-purified goat anti-dinitrophenyl bovine serum albumin IgG. After washing, peroxidase activity bound to the ball was assayed by fluorometry (the sandwich capture EIA I). In the sandwich capture EIA II, the complex was, after thorough washing, eluted from the ball with dinitrophenyl-L-lysine, and then peroxidase activity in the eluate was assayed. The thorough washing and elution processes improved the sensitivity 3.3-fold, and B substance in saliva samples from type B and AB secretors could be detected 200- to 500-fold more sensitively than hemagglutination inhibition, a method commonly used in forensic practices.
描述了夹心捕获酶免疫测定法（酶免疫测定法I和酶免疫测定法II）。水溶性B物质与亲和纯化的二硝基苯基山羊抗B IgG和亲和纯化的山羊抗B IgG-过氧化物酶结合物同时反应。由B物质、二硝基苯基IgG和IgG-过氧化物酶结合物形成的复合物被捕获到涂有亲和纯化的山羊抗二硝基苯基牛血清白蛋白IgG的聚苯乙烯球上。洗涤后，通过荧光法测定与球结合的过氧化物酶活性（夹心捕获酶免疫测定法I）。在夹心捕获酶免疫测定法II中，复合物在彻底洗涤后，用二硝基苯基-L-赖氨酸从球上洗脱，然后测定洗脱液中的过氧化物酶活性。彻底的洗涤和洗脱过程使灵敏度提高了3.3倍，并且B型和AB型分泌者唾液样本中的B物质检测灵敏度比法医实践中常用的血凝抑制法高200至500倍。
