Subject(s)
Antigens, Bacterial/analysis , Bordetella pertussis/growth & development , Adhesins, Bacterial/analysis , Antibodies, Monoclonal , Bacterial Outer Membrane Proteins/analysis , Bordetella pertussis/immunology , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Hemagglutinins/analysis , Virulence Factors, Bordetella/analysisABSTRACT
The anti-T cell monoclonal antibody (Mab) RIV9 (mouse IgG3, kappa) has been developed for clinical use in the treatment of allograft rejection. In order to obtain a clinical grade Mab preparation, RIV9 was purified from cell culture supernatants by protein A affinity and anion exchange chromatography. Reasonable yields of highly purified product could only be obtained if stabilising compounds were added and Tween 80 was used in all stages of the purification process. Prior to anion exchange chromatography, dextran sulphate (MW 5000) was added to keep the Mab in solution. Many other additives were tested but did not solubilise RIV9 under the low salt strength conditions required for ion exchange chromatography. The complex character of the solubility-determining factors was demonstrated by the influence of buffer composition, buffer concentration, pH, and sodium chloride concentration on the solubility of RIV9.
Subject(s)
Immunoglobulin G/isolation & purification , Animals , Antibodies, Monoclonal , Antigens, Differentiation, T-Lymphocyte/immunology , CD3 Complex , Chromatography, Affinity , Chromatography, Ion Exchange , Enzyme-Linked Immunosorbent Assay , Hybridomas , Immunoglobulin G/immunology , Mice , Osmolar Concentration , Receptors, Antigen, T-Cell/immunology , Solubility , T-Lymphocytes/immunologyABSTRACT
By testing two monoclonal antibodies (MoAb), we tried out two methods, which might be suitable for quality control of anti human T cell MoAb. RIV6 and RIV9 were investigated by flow cytometry and Enzyme-linked Immunosorbent Assay (ELISA). The characteristics, specificity and biological activity of RIV6 and RIV 9 were determined and compared respectively with other CD4 specific MoAb, OKT4, OKT4a, LEU3a and the CD3 specific MoAb OKT3 and LEU4 by flow cytometry. An ELISA was set up to quantify mouse IgG3 antibody. Specificity, plate variability and day to day effects were determined. The results illustrate that ELISA and flow cytometry are valuable tools in the quality control of these antibodies.
