PRODUCTION OF HUMAN ANTI-GLYCOPHORIN-A MONOCLONAL ANTIBODIES AND ITS PURIFICATION BY PSEUDOAFFINITY CHROMATOGRAPHY USING A CONVECTIVE INTERACTION MEDIA (CIM) MONOLITHIC COLUMN
B. Premkumar*, N. S. Jayaprakash and M. A. Vijayalakshmi
ABSTRACT
The group of red blood cell membrane glycoproteins known as glycophorins, in reference to their high carbohydrate content, have been used for years as a general model to probe the structure and function of membrane proteins and as specific markers of erythroid differentiation. The major glycoprotein of the human erythrocyte membrane, called glycophorin A (GPA), is a single polypeptide chain composed of 131 amino acid residues and approximately 125 sugar residues. GPA can act as receptor for malarial parasites, bacterial toxins and some viruses. This paper reports the production of anti-glycophorin A monoclonal antibodies (mAbs) and its purification using a convective interaction media (CIM) disk monolithic column. Hybridomas were generated by the fusion of mouse myeloma cell line (Sp2/0) and spleen cells from mouse immunized with Triton X-100 solubilized RBC membrane proteins. The hybridomas secreting antibodies specific for commercial glycophorin A were assayed by Enzyme Linked Immunosorbent Assay (ELISA) and the cloning of hybridoma cells was performed by limiting dilution method. The titers of the cell culture supernatant for the stable clones were found to be above 4096. The antibody produced by all the stable clones was found to be IgG1 with kappa (κ) light chain. Purification of IgG1 monoclonal antibodies from the supernatant of hybridoma cell culture was carried out by pseudobioaffinity chromatography using a CIM-Ethylene Diamine (EDA)-histidine disk. Higher purity of monoclonal antibody was obtained in the elution fractions of 25mM MOPS buffer, pH 6.5. The peak fractions eluted with 0.2M NaCl had a purification fold of about 8.27 and it showed good purity of IgG1 with higher specific activity based on SDS-PAGE analysis and ELISA. The results indicate that faster separation and efficient recovery of high purity anti-Glycophorin A mAbs could be achieved using CIM-EDA-histidine disk.
Keywords: Monoclonal antibody, human glycophorin A, CIM disk, pseudoaffinity, purification.
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