Objective: To prepare human cystatin C( CysC) recombinant protein and produce monoclonal antibodies with high affinity and specificity. Develop a competitive ELISA detection system to detect of CysC in human serum.
Methods: The CysC gene sequence was found on NCBI. The optimized gene fragments were synthesized and the recombinant CysC protein was expressed in Escherichia coli then used to immunize Balb/c mice. The positive hybridoma cell lines were obtained by hybridoma cell fusion techniques and ascites monoclonal antibody was prepared and purified. Affinity of the antibody was measured by indirect ELISA. Then competitive ELISA detection system was established, and 52 cases of human serum samples were detected by the detection system.
Results: Four stable cell lines secreting CysC monoclonal antibodies were obtained. Antibody Ab3 was used as a detection antibody and HRP labeling was performed. Its affinity constant was 4. 26 × 10~6L/mol. The linear range of detection was 0. 011-1. 924 μg/mL. The detection limit was 4. 598 ng/mL and IC_(50) was 0. 145 μg/mL. The established competitiveELISA serum detection system could accurately detect those 52 serum samples.
Conclusion: The monoclonal antibody against CysC with high affinity and specificity has been successfully obtained. A reliable competitive ELISA serum detection system is established. The method provides a basis for the development of CysC rapid immunoassay kit.
Keywords: competitive ELISA; human cystatin C; monoclonal antibody.