title: Production of human anti-glycophorin-A monoclonal antibodies and their purification by pseudoaffinity chromatography using a convective interaction media monolithic column.
abstract: 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.
authors.affiliation: Centre for Bio-separation Technology, Vellore Institute of Technology, VIT University, Vellore, Tamilnadu, India.
authors.forename: B Prem
authors.initials: BP
authors.lastname: Kumar
authors.forename: Poonam
authors.initials: P
authors.lastname: Rajak
authors.forename: M A
authors.initials: MA
authors.lastname: Vijayalakshmi
authors.forename: N S
authors.initials: NS
authors.lastname: Jayaprakash
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification*; D002845:Chromatography / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006021:Glycophorins / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000911:Antibodies, Monoclonal; D006021:Glycophorins