LARGE SCALE GENERATION OF MONOCLONAL ANTIBODY AGAINST EXTERACELLULAR PEPTIDE OF CD20 AND EVALUATION OF SPECIFIC BINDING TO CD20 EXPRESSING CELLS

Sineh Sepehr, K and Baradaran, B and Majidi, J and Abdolalizadeh, J (2014) LARGE SCALE GENERATION OF MONOCLONAL ANTIBODY AGAINST EXTERACELLULAR PEPTIDE OF CD20 AND EVALUATION OF SPECIFIC BINDING TO CD20 EXPRESSING CELLS. The Journal of Urmia University of Medical Sciences, 25 (4). pp. 363-372.

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Abstract

Nowadays, by advent of hybridoma technology in monoclonal antibodies production various cell markers could be evaluated in malignant and non-malignant cells. CD20, nonglycosylated phosphoprotein is as an ideal marker in leukemia and B-cell lymphoma diagnosis which is expressed on more than 95% of normal and neoplastic B-cells except for early B-cells and mature plasma. The prime aim of this study was to produce monoclonal antibody against CD20 and evaluation of specific binding to CD20 expressing cells. Materials & Methods: First, Balb/c mice were immunized 4 times with 100µg peptide from extracellular domain of CD20. Spleen cells of the most immune mouse were fused with SP2/0 by Poly Ethylene Glycol (PEG). The desired clones were selected for limiting dilution (L.D). Large scale of monoclonal antibodies was produced by ascetic fluid method. Monoclonal antibody was purified by Protein-A-Sepharose column affinity chromatography then confirmed by SDS-PAGE. Afterward, evaluation of specific binding of these antibodies was determined by immunological assay such as ELISA and Immunofluorescence and flowcytometry. Results: After cell fusion and screening, one desired monoclone achieved with absorbance about 2. Protein-A-Sepharose column affinity chromatography yielded about 5 mg of purified monoclonal antibody. The SDS-PAGE results confirmed purification of antibody. The result of ELISA, direct immunofluorescence staining and flow cytometry confirmed specific attachment to CD20. Conclusions: These results indicate that such monoclonal antibodies against CD20 can be used in diagnosis of CD20 in the cells surface by immunological assay such as ELISA and Immunofluorescence and flowcytometry

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