Kuhn H M, Basu S, Mayer H
Eur J Biochem. 1987 Jan 2;162(1):69-74. doi: 10.1111/j.1432-1033.1987.tb10543.x.
Enterobacterial common antigen (ECA) was isolated from a number of selected species (including Salmonella montevideo, Shigella sonnei and Plesiomonas shigelloides) using the extraction method described by Männel and Mayer [Eur. J. Biochem. 86, 361-370 (1978)]. ECA of all these species behaved identically in enzyme-linked immunosorption assay (ELISA) and in its inhibition using monoclonal anti-ECA antibodies. Immunoblotting showed a ladder-like pattern of at least 20 bands for all preparations tested. ECA modified at its lipid moiety (e.g. by phospholipases A2 and D or by mild acid hydrolysis) lost its coating capacity leaving, however, the serological reactivity as detected by inhibition assays intact. In contrast, reduction of the carboxylic groups of 2-acetamido-2-deoxy-D-mannopyranosyluronic acid destroyed the serological reactivity. Deacylated ECA was also not detectable in immunoblotting. Chemical reacylation restored the reactivity of deacylated ECA in ELISA and in immunoblot and thus proved the essential function of fatty acids for the physicochemical properties of the molecule. 2-Acetamido-2-deoxy-D-glucopyranose was identified as the reducing end of the ECA sugar chain after splitting off the lipid moiety by phospholipase D.
使用Männel和Mayer [《欧洲生物化学杂志》86, 361 - 370 (1978)]所描述的提取方法，从一些选定的菌种（包括蒙得维的亚沙门氏菌、宋内志贺氏菌和类志贺邻单胞菌）中分离出肠杆菌共同抗原（ECA）。所有这些菌种的ECA在酶联免疫吸附测定（ELISA）及其使用单克隆抗ECA抗体的抑制试验中表现相同。免疫印迹显示，所有测试制剂至少有20条带呈梯状模式。在其脂质部分被修饰的ECA（例如通过磷脂酶A2和D或轻度酸水解）失去了其包被能力，然而，抑制试验检测到的血清学反应性保持完整。相反，2-乙酰氨基-2-脱氧-D-甘露吡喃糖醛酸羧基的还原破坏了血清学反应性。在免疫印迹中也检测不到脱酰基的ECA。化学再酰化恢复了脱酰基ECA在ELISA和免疫印迹中的反应性，从而证明了脂肪酸对该分子物理化学性质的重要作用。在通过磷脂酶D去除脂质部分后，2-乙酰氨基-2-脱氧-D-葡萄糖被鉴定为ECA糖链的还原端。
