Preparation And Identification Of Mouse Anti-human IgE Monoclonal Antibody

ObjectiveThe current study is to investigate the preparation of mouse anti human IgE monoclonal antibody,and lay groundwork for the diagnosis of allergic diseases and development of proprietary humanized monoclonal anti-human IgE antibodies.MethodsIn this study,we first amplified the gene of IgE Fcε3-4 from the cDNA of CRL8033 cell line and cloned into pET32a(+)expression vector.This recombinant vector was transformed into BL21(DE3)E.coli and the recombinant protein expression was induced by IPTG.The antigen obtained from the inclusion body was renatured.The purity of the protein was assessed by SDS-PAGE.Finally we used the purified protein as an antigen to immunize BABL/c mice and the spleen cells were fused with SP2/0 after the third immunization.The hybridoma secreting monoclonal antibodies that could recognize human IgE was identified by ELISA,Western Blot and Immunofluorescence.Finally,the light and heavy chain variable regions of the anti-human IgE monoclonal antibody 3H4 and 6A10 were amplified by using ofuniversal primers and the amino acid sequences of the variable regions were analyzed by the protein prediction software.ResultsWe successfully constructed the pET32a(+)-IgE Fcε3-4 expression vector,purified the antigen from inclusion body,and used the purified protein as an antigen to immunize mice.Successfully prepared hybridoma cell lines secretes monoclonal antibodies capable of specifically recognize human IgE,which can be used for for ELISA,Western Blot and Immunofluorescence detection of human IgE.Finally,the amino acid sequence of the monoclonal antibody 3H4 and 6A10 variable regions were obtained by the protein prediction software.ConclusionA monoclonal antibody which can be used to specifically detect human IgE protein was obtained successfully,the foundation for detection of human IgE and development of proprietary humanized anti-human IgE monoclonal antibody was built.