Takeda T, Nair G B, Suzuki K, Zhe H X, Yokoo Y, De Mol P, Hemelhof W, Butzler J P, Takeda Y, Shimonishi Y
National Institute of Cholera and Enteric Diseases, Calcutta, India.
Infect Immun. 1993 Jan;61(1):289-94. doi: 10.1128/iai.61.1.289-294.1993.
A panel of monoclonal antibodies (MAbs) specific for the heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli was produced. All four MAbs (8G7, 53-4, 11C, and SH1) bound to native STh in an enzyme-linked immunosorbent assay to various degrees, with clone SH1 showing the best affinity. The MAbs were screened for neutralizing and guanylate cyclase-inhibiting activities by the suckling mouse assay and the cyclic GMP assay using T84 cells, respectively. The contact amino acid residues governing the reactivity of the four MAbs were precisely determined by using several chemically synthesized analogs of the various heat-stable enterotoxins (STa's). Three distinct antigenic sites of STh sufficiently removed from each other, one near the N terminus, another in the core functional region of the toxin, and the third in the C-terminal region, were recognized by the different MAbs. MAb SH1, which recognized Asn at position 4 and Tyr at position 5 from the N terminus was 100 times more potent in neutralizing the bioactivity of STh in the suckling mouse assay than was MAb 11C, which recognized Thr at position 16 and Tyr at position 19 from the N terminus of the STh molecule. The MAbs which recognized Leu at position 9 from the N terminus (MAb 53-4) and Tyr at position 19 from the N terminus (MAb 8G7) showed intermediate activities in the neutralization assay. The guanylate cyclase-inhibiting activities of SH1 and 11C essentially paralleled the results for the neutralization of bioactivity, while MAbs 53-4 and 8G7 exhibited reverse activity. These results indicate that MAbs that recognize the N-terminal residues which have been shown not to be essential for toxic activity have a potent protective capacity. None of the MAbs reacted with reduced and carboxy-methylated native STh. This suggests that all of the MAbs mediate their effect by reacting with conformation-dependent antigenic determinants.
制备了一组针对产肠毒素大肠杆菌热稳定肠毒素（STh）的单克隆抗体（MAb）。在酶联免疫吸附测定中，所有四种单克隆抗体（8G7、53-4、11C和SH1）均不同程度地与天然STh结合，其中克隆SH1表现出最佳亲和力。分别通过乳鼠试验和使用T84细胞的环鸟苷酸测定法筛选单克隆抗体的中和活性和鸟苷酸环化酶抑制活性。通过使用几种各种热稳定肠毒素（STa）的化学合成类似物，精确确定了决定四种单克隆抗体反应性的接触氨基酸残基。不同的单克隆抗体识别出STh的三个彼此充分分开的不同抗原位点，一个靠近N端，另一个在毒素的核心功能区域，第三个在C端区域。在乳鼠试验中，识别N端第4位的天冬酰胺和第5位的酪氨酸的单克隆抗体SH1中和STh生物活性的能力比识别STh分子N端第16位的苏氨酸和第19位的酪氨酸的单克隆抗体11C强100倍。识别N端第9位的亮氨酸的单克隆抗体（53-4）和识别N端第19位的酪氨酸的单克隆抗体（8G7）在中和试验中表现出中等活性。SH1和11C的鸟苷酸环化酶抑制活性与生物活性中和结果基本平行，而单克隆抗体53-4和8G7表现出相反的活性。这些结果表明，识别已显示对毒性活性非必需的N端残基的单克隆抗体具有强大的保护能力。没有一种单克隆抗体与还原和羧甲基化的天然STh反应。这表明所有单克隆抗体都是通过与构象依赖性抗原决定簇反应来介导其作用的。
