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首页> 外文期刊>Hybridoma >Production of human anti-glycophorin-a monoclonal antibodies and their purification by pseudoaffinity chromatography using a convective interaction media monolithic column
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Production of human anti-glycophorin-a monoclonal antibodies and their purification by pseudoaffinity chromatography using a convective interaction media monolithic column

机译：人抗糖原蛋白-a单克隆抗体的生产和对流相互作用介质整体柱的伪亲和色谱法纯化

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摘要

Monoclonal antibodies (MAbs) against glycophorin-A (GPA) could be used in identifying MN blood groups, detecting specific markers of erythroid differentiation, and studying parasite interactions. Large-scale production of MAbs in bioreactors demands an efficient and rapid separation technology. The present study describes the production of a human anti-GPA monoclonal antibody and its purification using a pseudo-bioaffinity L-histidine-convective interaction media (CIM) monolithic column. Hybridomas were generated by fusion of mouse myeloma cell line (Sp2/0) and spleen cells from the mouse immunized with Triton X-100 solubilized RBC membrane proteins. Hybridomas producing antibodies specific to commercial glycophorin-A were screened by indirect enzyme-linked immunosorbent assay (ELISA). The antibodies produced by the stable clones were found to be IgG1 with kappa light chain. Purification of IgG1 MAbs from the cell culture supernatant carried out with a CIM-EDA-histidine disk resulted in high specific activity with purification fold of 8.3 in the fraction eluted with MOPS buffer containing 0.2-M NaCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA showed that the antibodies obtained were highly pure, with high antigen-binding efficiency. The results indicate that faster separation and efficient recovery of high-purity anti-GPA MAbs could be achieved by using CIM-EDA-histidine disk.
机译：针对糖蛋白A（GPA）的单克隆抗体（MAb）可用于鉴定MN血型，检测红系分化的特定标记以及研究寄生虫的相互作用。在生物反应器中大规模生产单克隆抗体需要高效，快速的分离技术。本研究描述了人抗GPA单克隆抗体的生产及其使用假生物亲和性L-组氨酸-对流相互作用介质（CIM）整体柱的纯化。通过融合小鼠骨髓瘤细胞系（Sp2 / 0）和用Triton X-100增溶的RBC膜蛋白免疫的小鼠的脾细胞产生杂交瘤。通过间接酶联免疫吸附测定（ELISA）筛选产生对商业糖蛋白-A特异的抗体的杂交瘤。发现稳定克隆产生的抗体是具有κ轻链的IgG1。使用CIM-EDA-组氨酸圆盘从细胞培养上清液中纯化IgG1 MAb产生了高比活，在含有0.2-M NaCl的MOPS缓冲液洗脱的级分中，纯化倍数为8.3倍。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和酶联免疫吸附测定（ELISA）表明所获得的抗体纯度高，抗原结合效率高。结果表明，通过使用CIM-EDA-组氨酸盘可以实现高纯度抗GPA MAb的更快分离和有效回收。

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