Preparation and assessment of a monoclonal antibody against mouse NLRP3
Objective To prepare a monoclonal antibody(mAb)against mouse NOD-like receptor family pyrin domain-containing 3(NLRP3)and assess its specificity.Methods A gene fragment encoding mouse NLRP3 exon3(Ms-N3)was inserted into the vector p36-G3-throhFc to construct a recombinant plasmid named Ms-N3-throhFc.This plasmid was then transfected into HEK293F cells for eukaryotic expression.NLRP3-/-mice were immunized with Ms-N3 protein purified using a protein A chromatography column,and splenocytes from the immunized mice were fused with SP2/0 myeloma cells to generate hybridoma cells.Specific mAbs against murine NLRP3 from hybridoma cells were screened using ELISA and immunofluorescence assay(IFA).Results The Ms-N3-throhFc recombinant plasmid was successfully constructed and exhibited stable expression in HEK293F cells.Twelve hybridoma cell lines were initially screened using ELISA.IFA revealed that the mAb secreted by the 9-β8-3-2-C5 cell line specifically recognized the native form of mouse NLRP3 protein.The heavy and light chain subtypes of this mAb were identified as IgM and κ,respectively.Conclusion A monoclonal antibody against mouse NLRP3 has been successfully prepared.
NETL
NSTL
万方数据
