Generation of a novel high-affinity monoclonal antibody with conformational recognition epitope on human IgM.
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 (K(a) = 3.5 x 10(9)M(-1). Immunoblotting and competitive ELISA were done and the results showed that the harvested antibody recognizes a conformational epitope on the mu 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 IgG(1)kappa.
Sarikhani
Sina
S
Tarbiat Modares University, Tehran, Iran.
Mirshahi
Manouchehr
M
Gharaati
Mohammad Reza
MR
Mirshahi
Tooran
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D001709:Biotechnology / Q000379:methods*; D002459:Cell Fusion; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D015151:Immunoblotting; D007075:Immunoglobulin M / Q000737:chemistry* / Q000276:immunology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007075:Immunoglobulin M