Development of hybridoma cells producing monoclonal antibody against human epsilon (epsilon) chain.
Epsilon (epsilon) chain is one of the five classes of immunoglobulins that plays an important role in allergic diseases. Production of monoclonal antibodies by a single clonotype against different epitopes of epsilon chain have high priority in development of diagnostic kits. In this study, an attempt was made to produce monoclonal antibodies against human epsilon chain. Balb/c mice were immunized with semipurified epsilon chain and spleen cells were fused with Sp2/0 mouse myeloma cell line in the presence of poly ethylene glycol. Supernatant of hybridoma cells were screened for detection of antibody by Enzyme-linked Immuno Sorbent Assay (ELISA) method. Cloning of selective high absorbance wells were done with limiting dilution method. The suitable clones (monoclones) were selected by ELISA and confirmed by immunoblot. The subclasses of the chosen monoclonal antibodies were determined and the clones freezed and kept in liquid nitrogen. During this study three successful fusions were carried out, which resulted in over than 100 clones with high production of anti- epsilon chain. Twelve clones with the highest titers were selected for cloning. After limiting dilution more than 50 monoclonal antibodies were produced and the unsuitable one (C1F2) displayed higher absorbance in reaction with purified IgE, relatively high cross-reactivity with IgM, and the highest cross-reactivity with IgG. In Immunoblotting, presence of relatively high-density band in reaction with IgE was confirmed. The unsuitable monoclonal antibody was shown to be IgG1 subclass with kappa light chain. It seems that, this monoclonal antibody could not be successfully useful in diagnostic kits.
Majidi
J
J
Department of Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran. majidij@tbzmed.ac.ir
Hassan
Z M
ZM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006825:Hybridomas / Q000276:immunology*; D007136:Immunoglobulins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D007136:Immunoglobulins