Preparation And Application Of Momoclonal Antibody Specific To Estrogen Receptor(ERa) In Breast Cancer

Objective:In this work we cloned ERa gene and constructed the recombinant plasmid for expressing ERa protein.The purified recombinant proteins were prepared and then were used to immunize Balb/c mice.We prepared monoclonal antibodies against ERa and investaged its application on immunohistochemistry,which showed potential diagnostic value in immunohistochemistry.Methods:T47D cells with high expression of ERa were cultured for extracting total RNA.RT-PCR technology was used to amplify the ERa gene fragment,which was linked to the vector pET-27b(+).The recombinant plasmid was transformed into E.coli Rosetta.Recombinant proteins were induced by IPTG,identified by SDS-PAGE and purified by the chromatographic column containing Ni-NTA agarose resin.The purified ERa proteins were prepared according to antigen retrieval method with EDTA.The prepared ERa protein and normal ERa protein were then used to immunize Balb/c mices respectively.Hybridoma cells were prepared by cell fusion techniques.The monoclonal antibody was screened and evaluated by ELISA and immunohistochemistry techniques.The subtype of antibody were determined by ELISA using subtype identification kit.Ascites induced monoclonal antibody and purified by gravity column.Antibody purity was detected by SDS-PAGE.The diagnostic value of antibody was assessed using IHC assay.Results:1.The total RNA the recombinant plasmid pET-27b-ERa was constructed successfully.The ERa protein with a molecular weight about 27 kDa was expressed in a large amount induced by 0.5mM IPTG.2.The serum titers of Balb/c mice were more than 1.25 × 105 after 3 times of immunization.Antibody prepared by antigen treated with EDTA showed less nonspecific staining and more clear ERa nuclear localization detected with IHC.3.The ERa hybridoma cells were fused successfully.Immunohistochemistry assay of 2G10G4 and 2G10G12 showed the monoclonal antibody had a specific nuclear localization and with less background and non-specific staining.The purified antibody subtype was identified as IgGl,the light chain was ? type.Conclusions:The constructed ER? gene can be used successfully prepared antigen,The monoclonal antibody of ERa with single specificity and high affinity was prepared by immunizing mice and had a good performance on immunohistochemical staining of pathological tissue.The results showed that protein antigen prepared with EDTA retrieval was beneficial for the preparation of antibidy appling in immunohistochemistry.