McVie A, Ersfeld K, Rogan M T, Craig P S
Department of Biological Sciences, University of Salford, UK.
Acta Trop. 1997 Sep 15;67(1-2):19-35. doi: 10.1016/s0001-706x(97)00056-9.
A 165bp DNA fragment derived from the 12 kDa subunit of Echinococcus granulosus antigen B (AgB), a major hydatid cyst fluid antigen was cloned in the pMa1-c2 expression vector. A 52 kDa maltose binding-AgB fusion protein (rAgB.MBP) was produced and inclusion bodies containing the fusion protein were solubilised in urea and affinity purified on an amylose-Sepharose 6B column. The immunogenicity of the purified recombinant antigen for IgG4 antibody detection was tested with human serum using immunoblotting, ELISA and dot-ELISA assays and compared to native AgB. Both recombinant and native AgB preparations were highly reactive for human IgG4 antibodies in serum of cystic echinococcus (CE) patients. Recombinant AgB.MBP (rAgB.MBP) showed approximately 65% sensitivity in detection of IgG4 serum antibodies by ELISA from confirmed CE patients. Cross-reactivity (33%) occurred with alveolar echinococcosis (E. multilocularis) sera but recombinant AgB showed no seroreactivity with sera from other helminth infections tested (schistosomsis, onchocercsis, cysticercosis) or from uninfected individuals residing in CE endemic or non-endemic regions. The serologic sensitivity (63%) for IgG4 antibodies of a native AgB fraction enriched from human hydatid cyst fluid was similar to that for recombinant AgB (65%) though specificity was slightly lower (81%). A dot-ELISA for detection of total IgG, incorporating the rAgB.MBP resulted in 74% sensitivity and 88% specificity for human CE and 93% sensitivity and 65% specificity for native AgB. Recombinant AgB is a potential replacement for native antigens currently being used and could provide a better standardised E. granulosus specific test for clinical confirmation for CE especially for IgG4 antibody detection which appears to be predominantly associated with advanced disease.
从细粒棘球绦虫抗原B（AgB）12 kDa亚基衍生而来的一段165 bp DNA片段被克隆到pMa1-c2表达载体中，AgB是一种主要的包虫囊肿液抗原。产生了一种52 kDa的麦芽糖结合-AgB融合蛋白（rAgB.MBP），含有该融合蛋白的包涵体用尿素溶解，并在直链淀粉-琼脂糖6B柱上进行亲和纯化。使用免疫印迹、ELISA和斑点ELISA试验，用人血清检测纯化重组抗原对IgG4抗体检测的免疫原性，并与天然AgB进行比较。重组和天然AgB制剂对囊性棘球蚴病（CE）患者血清中的人IgG4抗体均具有高反应性。重组AgB.MBP（rAgB.MBP）通过ELISA检测确诊CE患者IgG4血清抗体的敏感性约为65%。与肺泡棘球蚴病（多房棘球绦虫）血清发生交叉反应（33%），但重组AgB与其他测试的蠕虫感染（血吸虫病、盘尾丝虫病、囊尾蚴病）血清或来自CE流行或非流行地区的未感染个体的血清无血清反应性。从人包虫囊肿液中富集的天然AgB组分对IgG4抗体的血清学敏感性（63%）与重组AgB相似（65%），尽管特异性略低（81%）。采用rAgB.MBP的检测总IgG的斑点ELISA对人CE的敏感性为74%，特异性为88%，对天然AgB的敏感性为93%，特异性为65%。重组AgB是目前正在使用的天然抗原的潜在替代品，可为CE的临床确诊提供更好的标准化细粒棘球绦虫特异性检测，尤其是对于IgG4抗体检测，IgG4抗体检测似乎主要与晚期疾病相关。
