人IL-35单克隆抗体制备及其在定量ELISA检测方法中的应用

Preparation of monoclonal antibodies against human interleukin-35 and their application in a quantitative ELISA for interleukin-35 detection

摘要:

为了建立人白细胞介素-35 (IL-35) 的定量ELISA检测方法，RT-PCR 克隆了人IL-35的IL-27EBI3亚基和IL-12p35亚基的编码基因，在大肠杆菌中实现了2个亚基的高效表达。以大肠杆菌表达的IL-27EBI3和IL-12p35重组蛋白作为免疫原，免疫BaLb/c小鼠，选取阳性血清小鼠的脾细胞与小鼠骨髓瘤SP2/0细胞融合，用间接ELISA和有限稀释法进行单克隆杂交瘤细胞的筛选和克隆，获得了稳定分泌抗IL-27EBI3和抗IL-12p35单克隆抗体的杂交瘤细胞株。在效价测定和特异性鉴定的基础上，进一步筛选出一株能稳定分泌抗IL-27EBI3单克隆抗体的杂交瘤细胞株3B11和一株能稳定分泌抗IL-12p35 单克隆抗体杂交瘤细胞株3A10，亚类鉴定两株单抗均为IgG1。应用单抗3B11和3A10建立了IL-35双抗夹心定量ELISA检测方法，借助生物素-亲和素放大效应，该方法检测IL-35线性范围为3.12–200 pg/mL，最低检测限为1.26 pg/mL，与多种其他抗原的交叉反应率为0.1%，批内相对标准偏差 (RSD) 为5.1%–5.6%，批间RSD为5.6%–7.2%，添加回收率为89%–103%，达到定量分析的要求，为进一步组装IL-35定量ELISA检测试剂盒打下了基础。

Abstract:

To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12–200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%–5.6% and 5.6%–7.2%, respectively, and the fortified recovery was 89%–103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.