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

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