Jiang L, Xu X-N, Li X, Xue H-C, Feng Z
National Institute of Parasitic Diseases, Chinese Centre for Disease Control and Prevention, Shanghai 200025, China.
Parasite Immunol. 2004 Oct;26(10):377-85. doi: 10.1111/j.0141-9838.2004.00723.x.
The aim of this work was to identify the immunodominant regions of the Em18 antigen to improve the specificity in diagnosis of alveolar echinococcosis (AE). Two recombinant antigens ReEm18-1 and ReEm18-2, which have the same sequence except that nine amino acid residues are absent in ReEm18-2, were tested by ELISA and Western Blot (WB) for their diagnostic efficiency. Serological evaluation of the two antigens demonstrated that the sensitivity of both antigens was 95.5% in ELISA and WB, and the specificity was 93.6% and 95.7% in ELISA, and 81.4% and 82.9% in WB, respectively. Five more expression clones (EmS1-EmS5), which contain different regions of the Em18 sequence, were constructed for defining the immunodominant regions of the antigen. Fourteen monoclonal antibodies (mAbs) against ReEm18-2 antigen and the sera from different groups of patients were used to identify the epitope regions of the five antigen fragments. Results showed that the epitopes recognized by the mAbs are located in the N-terminal third of the sequence, but the immunodominant area recognized by native serum antibodies may be located further downstream (C-terminal) in the sequence. The nonspecific cross-reactivity is due to epitopes present in the C-terminal third of the sequence. The antigen fragments that contain the first two-thirds of the sequence have the same sensitivity to AE sera as those of the ReEm18-1 and ReEm18-2 antigens, but removal of the C-terminal third of the sequence improved the specificity of the assay from 93.6% to 99.3% (ELISA) and 81.4% to 90.7% (WB). We conclude that the necessary part of the ReEm18 antigen sequence for AE diagnosis is the N-terminal half to two-thirds of the entire sequence.
本研究旨在确定Em18抗原的免疫显性区域，以提高肺泡型包虫病（AE）诊断的特异性。通过ELISA和Western Blot（WB）检测了两种重组抗原ReEm18-1和ReEm18-2的诊断效率，这两种抗原除ReEm18-2缺少九个氨基酸残基外序列相同。对这两种抗原的血清学评估表明，两种抗原在ELISA和WB中的敏感性均为95.5%，在ELISA中的特异性分别为93.6%和95.7%，在WB中分别为81.4%和82.9%。构建了另外五个包含Em18序列不同区域的表达克隆（EmS1-EmS5），以确定该抗原的免疫显性区域。使用针对ReEm18-2抗原的14种单克隆抗体（mAb）和不同组患者的血清来鉴定五个抗原片段的表位区域。结果表明，mAb识别的表位位于序列的N端三分之一处，但天然血清抗体识别的免疫显性区域可能位于序列下游更远的位置（C端）。非特异性交叉反应是由于序列C端三分之一处存在的表位。包含序列前三分之二的抗原片段对AE血清的敏感性与ReEm18-1和ReEm18-2抗原相同，但去除序列的C端三分之一可将检测的特异性从93.6%提高到99.3%（ELISA），从81.4%提高到90.7%（WB）。我们得出结论，AE诊断所需ReEm18抗原序列的必要部分是整个序列的N端一半至三分之二。
