Germani Y, Guesdon J L, Begaud E, Moreau J P
Unité de Bactériologie expérimentale, Institut Pasteur, Nouméa.
Ann Inst Pasteur Microbiol. 1987 Nov-Dec;138(6):681-92. doi: 10.1016/0769-2609(87)90146-3.
Two simple two-step competitive enzyme-linked immunoassays for human E. coli heat-labile enterotoxin (LTh) employing microtitration plates coated with rabbit anti-LTh antibody (ELISA) or GM1 ganglioside (GM1-ELISA) are described. LTh of the test sample competed with the same toxin coupled with horse-radish peroxidase. ELISA and GM1-ELISA were able to detect, respectively, as low as 5 ng and 6.5 ng TLh/ml and up to 9 and 11 micrograms TLh/ml. Both techniques were applied to the study of 167 infant diarrhoeas; ETEC producing LTh were identified in 17 diarrhoeal stools. When the faeces were diluted in phosphate buffer, only 17.6% (3 stools) and 29% (5 stools) of LTh-positive faeces were identified in ELISA and GM1-ELISA. When the stools were diluted with phenylmethylsulphonyl fluoride (PMSF), a synthetic protease inhibitor, 82% (14 stools) and 88% (15 stools) of LTh-positive stool supernatants were detected. Aprotinin, another protease inhibitor, was without effect and foetal calf serum, horse serum and bovine serum albumin enabled detection of only a low percentage of LTh-positive stools.
本文描述了两种简单的两步竞争性酶联免疫分析法，用于检测人源大肠杆菌不耐热肠毒素（LTh）。这两种方法分别采用包被有兔抗LTh抗体的微量滴定板（ELISA法）或GM1神经节苷脂（GM1-ELISA法）。测试样品中的LTh与辣根过氧化物酶偶联的相同毒素竞争。ELISA法和GM1-ELISA法分别能够检测低至5 ng和6.5 ng TLh/ml的LTh，检测上限分别为9 μg和11 μg TLh/ml。这两种技术都应用于167例婴儿腹泻的研究；在17份腹泻粪便中鉴定出产生LTh的肠毒素大肠杆菌（ETEC）。当粪便用磷酸盐缓冲液稀释时，ELISA法和GM1-ELISA法分别仅能鉴定出17.6%（3份粪便）和29%（5份粪便）的LTh阳性粪便。当粪便用合成蛋白酶抑制剂苯甲基磺酰氟（PMSF）稀释时，分别检测到82%（14份粪便）和88%（15份粪便）的LTh阳性粪便上清液。另一种蛋白酶抑制剂抑肽酶无效，而胎牛血清、马血清和牛血清白蛋白仅能检测到低比例的LTh阳性粪便。
