A dual monoclonal antibody sandwich enzyme-linked immunosorbent assay (mAb sandwich ELISA) has been developed using rabbit monoclonal antibodies generated by Ecobody technology, which includes the isolation of single B cells binding to a specific antigen, amplification of the heavy and light chains of these immunoglobulins, and expression of the fragment of antigen binding (Fab) by cell-free protein synthesis (CFPS). A rabbit was immunized with swine influenza virus (SIV) vaccine, from which single B cells binding to the antigen were isolated. Then, immunoglobulin mRNA was amplified from single cells by reverse transcription-polymerase chain reaction, followed by the attachment of a T7 promoter, appropriate tags, and a T7 terminator for the expression of the Fab portion by CFPS. By taking advantage of two different peptide tags fused to the same Fab, optimal combinations for coating Fab on assay plates and detecting Fab, both synthesized by CFPS, were investigated for mAb sandwich ELISA. Pairs of Fab detected 0.5 ng SIV in the assay. In summary, this result showed the applicability of Ecobody technology for a variety of immunodetection kits for high throughput analyses.

已使用由Ecobody技术生成的兔单克隆抗体开发了一种双单克隆抗体三明治酶联免疫吸附测定（mAb夹心ELISA），包括分离与特定抗原结合的单个B细胞，扩增这些抗体的重链和轻链免疫球蛋白，并通过无细胞蛋白质合成（CFPS）表达抗原结合片段（Fab）。用猪流感病毒（SIV）疫苗免疫兔子，从中分离出与抗原结合的单个B细胞。然后，通过逆转录-聚合酶链反应从单个细胞扩增免疫球蛋白mRNA，然后通过CFPS附着T7启动子，合适的标签和T7终止子以表达Fab部分。通过利用与同一Fab融合的两种不同的肽标签，研究了通过CFPS合成的将Fab包被在测定板上和检测Fab的最佳组合，用于mAb夹心ELISA。成对的Fab在测定中检测到0.5 ng SIV。总而言之，该结果表明Ecobody技术可用于多种用于高通量分析的免疫检测试剂盒。