Main content area

Generation of a Novel High-Affinity Monoclonal Antibody with Conformational Recognition Epitope on Human IgM

Sarikhani, Sina, Mirshahi, Manouchehr, Gharaati, Mohammad Reza, Mirshahi, Tooran
Applied biochemistry and biotechnology 2010 v.162 no.5 pp. 1249-1257
agarose, antigens, blood, cross reaction, enzyme-linked immunosorbent assay, humans, hybridomas, hybrids, immunoblotting, immunoglobulin M, ion exchange chromatography, isotypes, mice, polyacrylamide gel electrophoresis, polyethylene glycol, splenocytes
As IgM is the first isotype of antibody which appears in blood after initial exposure to a foreign antigen in the pattern of primary response, detection, and quantification of this molecule in blood seems invaluable. To approach these goals, generation, and characterization of a highly specific mAb (monoclonal antibody) against human IgM were investigated. Human IgM immunoglobulins were used to immunize Balb/c mice. Spleen cells taken from the immunized animals were fused with SP2/O myeloma cells using PEG (polyethylene glycol, MW 1450) as fusogen. The hybridomas were cultured in HAT containing medium and supernatants from the growing hybrids were screened by enzyme-linked immunosorbent assay (ELISA) using plates coated with pure human IgM and the positive wells were then cloned at limiting dilutions. The best clone designated as MAN-1, was injected intraperitoneally to some Pristane-injected mice. Anti-IgM mAb was purified from the animals' ascitic fluid by protein-G sepharose followed by DEAE-cellulose ion exchange chromatography. MAN-1 interacted with human IgM with a very high specificity and affinity. The purity of the sample was tested by SDS-PAGE and the affinity constant was measured [graphic removed] . Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the μ chain of human IgM and there was no cross-reactivity with other subclasses of immunoglobulins. Furthermore, isotyping test was done and the results showed the subclass of the obtained mAb which was IgG₁κ.