The Preparation Of Antibody Against Human Cystatin C, And Establishment Of Immunoassay For Human Cystatin C And Evaluation On Methodology

Cystatin C, a member of the cystatin super-family of cysteine proteinase inhibitors, has been shown to be a novel renal functional assessment index with more specificity and sensitivity than traditional indices of serum urea, uric acid and creatinine, as well as endogenous creatinine clearance rate (Ccr). Food and Drug Administration of USA published 26 latest promising determination items in laboratory medicines field on webpage in 2002, and Cys C was one of them. However, the up-to-date determination of serum Cys C in clinical laboratories is based on expensive import equipments and immunoassay kits so that the popularization of this valuable indicator of glomerular filtration function is restricted in China and other developing countries.Objective:To construct a prokaryotic expression vector of Cys C, to purify and identify Cys C protein produced by the expression system and to prepare its antiserum after rabbits were immunized with the fusion protein in order to provide a basis of establishment of clinical immunoassays for human serum Cys C. With automatic biochemical analyzer and self-made antibody, particle enhanced turbidimetric immunoassay method for human serum Cys C was established and methologically evaluated, in an effort to settle a foundation for its clinical application. Furthermore, Balb/c mice were immunized with the recombinant protein, and the monoclonal antibody was produced, which could be used to establish other immunoassay methods, and to explore deeply the biological roles and mechanism of Cys C.Methods:1. Total RNA was isolated from HL-60 cell line, and human Cys C gene was amplified with RT-PCR. The cDNA fragment was cloned into pET32a(+) prokaryotic expression vector.2. The vectors were transformed into E.coli BL 21(DE3) in which Cys C expression was induced, and the protein was purified through Ni2+ affinity chromatography.3. The antiserum was prepared after rabbits were immunized with the fusion protein.4. The purified antibody was covalently attached to the uniform microparticles, a particle enhanced turbidimetric immunoassay method for human serum Cys C was established, and evaluation tests of methodology were done, including recovery test, repeatability test, linear range investigation, interference test, as well as comparison test with commercial import kits.5. Mice spleen cells and myeloma cells (SP2/0) were fused after Balb/c mice were immunized with the recombinant protein. By the way of cloning and screening, the hybridoma cell lines were established, with which large-scale monoclonal antibodies produced in mice ascites was prepared.Results:1. It was shown that the expressed Cys C fusion protein was about 35 kD, mainly presented in the inclusion body of E.coli, which could be purified through Ni2+ affinity chromatography. Antiserum was prepared after rabbits were immunized with the purified protein.2. A particle enhanced turbidimetric immunoassay method for human serum Cys C was established. The comparison test revealed that the performance of self-established immunoassay method was equal to the import kits. The results of evaluation tests of methodology also showed that it was a rapid, sensitive, accurate and automated immunoassay that was applicable in clinical laboratory.3. Two hybridoma cell lines were achieved successfully with hybridoma technique, which could stably secret specific McAb against Cys C. After one of the hybridoma cell lines was injected into mice abdominal cavity, the ascites abundant in McAb was obtained. Conclusions:1. Cys C fusion protein is obtained by the method of genetic recombination technique, and rabbit antiserum (polyclonal antibody) against human Cys C is produced. Besides that, monoclonal antibody against human Cys C is prepared.2. With self-prepared polyclonal antibody, a particle enhanced turbidimetric immunoassay method for human serum Cys C is successfully established, which could be applied in most clinical laboratories with automatic biochemical analyzer extensively equipped. It should prompt the clinical application of serum Cys C test as the marker of renal glomerular filtration function.