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

Author:
Sarikhani, Sina, Mirshahi, Manouchehr, Gharaati, Mohammad Reza, Mirshahi, Tooran
Source:
Applied biochemistry and biotechnology 2010 v.162 no.5 pp. 1249-1257
ISSN:
0273-2289
Subject:
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
Abstract:
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.
Agid:
640840