Establishment of a sandwich ELISA method for detection of matrix metalloproteinase 9 protein in sera samples and preliminary application for hepatocellular carcinoma patients / 中华检验医学杂志

ABSTRACT

Objective Expression of recombinant human matrix metalloproteinase 9 (MMP9) protein in E coli,To establish a sandwich ELISA method for detecting MMP9 in sera and analyses of the difference of serum MMP9 in health population and the patient with hepatocellular carcinoma (HCC). Methods A fragment MMP9 sequence from nt 721-1 156 was amplified with PCR from the synthesized cDNAs of human liver tissues and inserted into the prokaryotic-expression plasmid pQE30.HIS-MMP9 fusion protein was expressed and purified from E coli MIS cells.The specific antibodies were elicited in rabbits and guinea pigs by immunization of the purified HIS-MMP9.After purifing antibodies with CNBr-activated Sepharose 4B,a sandwich ELISA technique was established.The serum MMP9 proteins were evaluated in 227 health adults and 193 HCC patients.Results SDS-PAGE displayed that the molecular-weight of the expressed fusion protein was about 17 000.The prepared antisera were able to recognize both recombinant and endogenously expressed MMP9 from human neutrophil and HepG2 cells.It was found that the average level of MMP9 proteins in HCC patients was higher than that in health control,showing significantly statistic difference.Conclusions The established method could reflect the level of serum MMPg.The data in this study supply scientific basis of generating methodology for screening metastasis and reoccurrence of HCC, using serum MMP9 as index.