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 \( \left( {{K_{\text{a}}} = {\text{3}}.{\text{5}} \times {\text{1}}{0^{\text{9}}}{{\text{M}}^{{\text{ - 1}}}}} \right) \). 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.
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This work was financially supported by the Monoclonal Antibodies Research Centre, Simaye Iran street Tehran, Iran and all rights are reserved to this company.
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Sarikhani, S., Mirshahi, M., Gharaati, M.R. et al. Generation of a Novel High-Affinity Monoclonal Antibody with Conformational Recognition Epitope on Human IgM. Appl Biochem Biotechnol 162, 1249–1257 (2010). https://doi.org/10.1007/s12010-009-8873-8
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DOI: https://doi.org/10.1007/s12010-009-8873-8