Development of a monoclonal antibody-based sandwich ELISA for detection of guinea pig interleukin-2
Development of a monoclonal antibody-based sandwich ELISA for detection of guinea pig interleukin-2
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DOI：
10.1292/jvms.68.1281
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发表时间：
2006-12-01
影响因子：
1.2
通讯作者：
Iwata, Hiroyuki
中科院分区：
文献类型：
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作者：
Adachi, Satoshi;Hashimoto, Tomomi;Iwata, Hiroyuki
白细胞介素2 （Interleukin 2, IL-2）是Th0和th1型辅助性T细胞释放的一种T细胞增殖因子，是免疫应答的重要细胞因子。本研究利用大肠杆菌制备了重组谷胱甘肽s -转移酶(GST)-豚鼠IL-2 （gil -2）融合蛋白，并以该蛋白为免疫原，制备了抗gil -2的单克隆抗体，为豚鼠免疫应答研究奠定了基础。建立了三个稳定的杂杂瘤细胞系，并通过酶联免疫吸附试验（ELISA）和/或免疫印迹分析显示，每个单抗与大肠杆菌和感染重组杆状病毒的昆虫细胞产生的重组GPIL-2具有特异性结合。同型分析显示，这3个单克隆抗体均为IgG1，并具有kappa链。此外，通过竞争结合试验对它们的表位进行评估表明，本研究获得的单克隆抗体结合了三个不同的表位。因此，基于GPIL-2不同表位特异性的两种单克隆抗体，建立了检测GPIL-2的夹心ELISA，其灵敏度阈值约为0.3 ng/ml GPIL-2。
Interleukin 2 (IL-2) is a T cell proliferation factor released by Th0- and Th1-type helper T cells and is an essential cytokine for immune responses. In the present study, recombinant glutathione S-transferase (GST)-guinea pig IL-2 (GPIL-2) fusion protein was prepared by Escherichia coli (E. coli) and by using this protein as an immunogen, monoclonal antibodies (mAbs) against GPIL-2 were produced to establish a basis for a research on immune responses in guinea pigs. Three stable hybridoma cell lines were established, and specific binding of each mAb to recombinant GPIL-2 produced by E. coli and insect cells infected with recombinant baculovirus was shown by enzyme linked immunosorbent assay (ELISA) and/or immunoblot analysis. Isotype analyses of these mAbs revealed that all three mAbs were IgG1 and had kappa chain. Furthermore, assessment of their epitopes by competition binding assay indicated that the mAbs obtained in this study bound to three different epitopes. Thus, a sandwich ELISA based on the two mAbs specific to different GPIL-2 epitopes was developed for detection of GPIL-2, which had a sensitivity threshold of about 0.3 ng/ml of GPIL-2.