Development Of Antibody For Immunohistochemical Detection Of Programmed Death Receptor PD-1

Objective:Programmed cell death-1(PD-1),an inducible immune modulatory receptor,has emerged as a promising clinical strategy for the treatment of cancer.In this study,we cloned,expressed and purified the recombinant PD-1.Furthermore,an anti-PD-1 high-affinity monoclonal antibody was prepared using hybridoma technique.We prepared anti-PD-1 monoclonal antibody and investigated its application on immunohistochemistry,which showed potential diagnostic value in research and immunohistochemistry.Methods:The full-length gene fragment of PD-1 was synthesized by Nanjing Kingsi for.In this study,the PD-1 gene was cloned and inserted into the expression plasmid pET-27b(+)and pET-22b(+).The recombinant plasmids pET-27b(＋)-PD-1 and pET-22b(+)-PD-1 were confirmed by restriction enzymes and sequencing analysis,then transformed into Escherichia coli BL21(DE3).The fused protein was expressed by induction of 0.5mM IPTG and purified by Ni2+ affinity chromatography.Furthermore,an anti-PD-1 high-affinity monoclonal antibody was prepared using hybridoma technique and analyzed by immunohistochemistry technology.Results:1.1.We had successfully cloned,expressed and purified PD-1 protein.High purity PD-1 was obtained from BL21(DE3)cells.2.An anti-PD-1 high-affinity monoclonal antibody 9C12-C8 was prepared using hybridoma technique.The purified antibody subtype was identified as IgG2a,the light chain was k subtype.3.IHC assay showed the monoclonal antibody had a specific localization on membrane and less background and non-specific staining.Besides,western-blot results also showed excellent specificity.Conclusions:The anti-PD-1 high-affinity monoclonal antibody which is prepared in this study could successfully obtain high specificity,affinity and have good effect in immunohistochemistry.