Production and characterization of monoclonal antibodies against human IgG in Balb/c mouse.

摘要

Immunoglobulin G (IgG) is the main immunoglobulin in natural human serum. It constitutes 70 to 75 percent of all immunoglobulins. Monoclonal antibodies have many applications in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgG are used in most diagnostic kits. For production of monoclonal antibodies against human IgG, spleen cells of the most immune mouse were fused with SP2/0 (myeloma cells) using Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Then, the supernatant of suitable monoclones were assessed for cross reactivity with IgM & IgA by ELISA and confirmed by immunobloting. The subclasses of the selected monoclonal antibodies were determined and the clones were frozen and kept in liquid nitrogen. Finally, suitable monoclone was injected into the mouse, intraperitoneally, that has been primed with Pristane. In this study, 127 clones were obtained of which 15 clones had absorbance more than 1 which two of them with absorbance about 1.5 were selected for limiting dilution. The yield of limiting dilution was 6 clones with absorbance about 1.8 which did not show cross reactivity with IgM & IgA. Among these clones, G2 monoclone with IgG1 subclass was selected as suitable one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the suitable clone was implanted in the peritoneum of the Balb/c mouse and its titer was measured, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgA. Monoclonal antibody was purified by chromatography, confirmed by SDS- PAGE, conjugated with enzyme and applied for diagnostic kits.