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首页> 美国卫生研究院文献>Clinical and Diagnostic Laboratory Immunology >An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs.
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An indirect double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using baculovirus-expressed antigen for the detection of antibodies to glycoprotein E of pseudorabies virus and comparison of the method with blocking ELISAs.

机译：使用杆状病毒表达的抗原的间接双抗体夹心酶联免疫吸附测定（ELISA）用于检测假狂犬病病毒糖蛋白E抗体并与封闭ELISA进行比较。

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Antibodies in porcine sera against glycoprotein E (gE) of pseudorabies virus (PRV) are usually measured in blocking enzyme-linked immunosorbent assays (ELISAs) with one or two murine monoclonal antibodies (MAbs) directed against gE. Our aim was to develop a confirmation assay which is based on another principle and which is able to detect antibodies directed against most potential binding sites on gE with high specificity. Therefore, we developed an indirect double-antibody sandwich assay (IDAS) using recombinant gE expressed by baculovirus (BacgE960). A fragment of the gE gene consisting of nucleotide positions +60 to +1020 of gE, coding for the major antigenic sites of gE but not the transmembrane region, was cloned behind the signal sequence of PRV gG and the p10 promoter in a baculovirus vector. Immunoblot analysis showed that the expressed protein reacted with MAbs directed against five of the six antigenic sites on gE. Although the conformation of some antigenic sites, notably antigenic sites E and C, was not identical to their natural conformation, the expressed protein bound gE-specific antibodies in porcine sera in Western blots (immunoblots) and ELISAs. For the IDAS, a coating MAb directed against the nonimmunodominant antigenic site A on gE was chosen. A major obstacle in binding ELISAs, such as the IDAS, appeared to be the high nonspecific binding activity observed in porcine sera. As a result, sera could be tested only in relatively high dilutions in the BacgE960 IDAS, in contrast to the testing of sera in blocking ELISAs. The sensitivity and specificity of the newly developed BacgE960 IDAS were evaluated and compared with those of five commercially available blocking ELISAs by using several sets of sera of known PRV disease history. The BacgE960 IDAS assay had a high diagnostic specificity and a moderate sensitivity. The five blocking ELISAs differed remarkably in sensitivity and specificity, thereby illustrating the need for standardization and confirmation. We conclude that the BacgE960 IDAS is a useful and specific additional (confirmatory) test for the detection of antibodies to gE.
机译：猪血清中针对伪狂犬病病毒（PRV）的糖蛋白E（gE）的抗体通常在封闭酶联免疫吸附测定（ELISA）中用一种或两种针对gE的鼠单克隆抗体（MAb）进行测量。我们的目的是开发基于另一原理的确认分析，该分析能够以高特异性检测针对gE上大多数潜在结合位点的抗体。因此，我们开发了使用杆状病毒（BacgE960）表达的重组gE的间接双抗体夹心测定（IDAS）。在杆状病毒载体中，将由gE的核苷酸位置+60到+1020组成的gE基因的片段克隆到PRV gG的信号序列和p10启动子的后面，该片段编码gE的主要抗原位点而不是跨膜区域。免疫印迹分析表明，表达的蛋白质与针对gE上六个抗原位点中的五个的单克隆抗体反应。尽管一些抗原性位点的构象，特别是抗原性位点E和C，与它们的天然构象不同，但在Western印迹（免疫印迹）和ELISA中，表达的蛋白结合了猪血清中的gE特异性抗体。对于IDAS，选择了针对gE上非免疫缺陷抗原位点A的包被单抗。结合ELISA（例如IDAS）的主要障碍似乎是在猪血清中观察到的高非特异性结合活性。结果，与封闭ELISA中的血清测试相比，只能在BacgE960 IDAS中以相对较高的稀释度测试血清。通过使用几套已知的PRV病史的血清，评估了新开发的BacgE960 IDAS的敏感性和特异性，并将其与五种市售封闭ELISA的敏感性和特异性进行了比较。 BacgE960 IDAS分析具有很高的诊断特异性和中等灵敏度。五个封闭ELISA的敏感性和特异性显着不同，从而说明了标准化和确认的必要性。我们得出结论，BacgE960 IDAS是用于检测gE抗体的有用且特定的附加（确认）测试。

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