Study of the separation of mouse monoclonal antibodies by pseudobioaffinity chromatography using matrix-linked histidine and histamine.
The selective retention of proteins on matrix-linked histidine has been shown to depend on chromatographic conditions: pH, temperature and ionic strength. An extension of this study to separate mouse monoclonal antibodies on histidyl-Sepharose is presented here; the roles of different functional groups such as imidazole, primary amine and carboxyl groups are elucidated by using histamine-Sepharose and histidine linked via the carboxyl group of the alpha-amino acid. We separated two monoclonal antibodies, immunoglobulin G1 (IgG1) from a culture supernatant and IgG2b from ascites fluid precipitated with 50% ammonium sulphate. The pseudoselective retention of monoclonal IgG1 on the three different matrices and IgG2b on histidyl-aminohexyl-Sepharose was achieved at pH 7.4. The purity of the final monoclonal antibody preparation determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions proved the separation of the monoclonal antibodies (IgG1, IgG2b) from other contaminating proteins such as albumin and transferrin. Quantitation of the mouse monoclonal antibodies was carried out using enzyme-linked immunosorbent assay.
el-Kak
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Laboratoire de Technologie des Séparations, Université de Technologie de Compiègne, France.
Vijayalakshmi
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MA
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification*; D001201:Ascites / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006632:Histamine / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006825:Hybridomas; D051379:Mice
D000911:Antibodies, Monoclonal; D006639:Histidine; D006632:Histamine