Objective: To prepare monoclonal antibodies (mAbs) against S100A9, and characterize these antibodies' properties for functional studies.
Methods: We amplified cDNA fragment of S100A9 from adult human liver sample, then constructed the vectors pGEX-4T-1-S100A9 and pET-32a-S100A9 for protein expression. The S100A9 protein fused respectively with the His-tag and GST-tag were expressed in E.coli and purified for immunizing mice as antigen and hybridoma screening, respectively. After hybriodma screening, the titer of mAbs was determined by ELISA and the specificity was identified by Western blotting and indirect immunofluorescence technique.
Results: Both GST-tagged and His-tagged S100A9 fusion proteins were successfully constructed and expressed in E.coli. Eighteen hybridoma cell strains secreting S100A9 mAbs were obtained, of which 15 showed strong positive reaction but 3 showed weak positive reaction to the recombinant protein in Western blotting. Two hybridoma cell strains were capable of binding to S100A9 native protein in tumor tissue interstitial fluid.
Conclusion: We successfully prepared the mAbs against S100A9 protein, which provide the useful tool for further study on biological function and clinical detection of S100A9.