Preparation Of Monoclonal Antibody Against Vitronectin And Its Primary Application In Clinical Examination

Background and Objective:Hepatocellular carcinoma (HCC) is one of the most common malignant cancers in China. Because of its difficulty in early stage diagnosis the rapid progress, there is no effective precaution and treatment measures for HCC. Therefore, it is of great significance to persue new molecular markers of liver cancer, develop clinical detection reagents and translate scientific research results into clinical products. Vitronectin as a potential disease marker of liver cancer was found in recent years. Research shows that the the expression level of VN is correlated with different stages of liver cancer, indicating that it is essential to study the relationship between VN and liver cancer exploiting highly specific antibodies against VN. Currently, there is no high quality antibodies and Clinical kit for VN protein. This study aims to develop proprietary VN monoclonal antibodies (mAbs), especially paired antibodies, and explore the possibility of providing the raw material for clinical diagnostic test kit based on sandwich ELISA.Methods:His-VN recombinant protein highly expressed in E. coli was purified and immunized with ajuvant Quick Atibody-Mouse 5W into Balb/c mice for preparing monoclonal antibodies. Hybridoma cells were screened by ELISA and Western blot. The positive mAbs were further determined whether they could be match-paired utilizing sandwich ELISA assays of serum VN and recombinant VN, and Fortebio BLI assays of recombinant VN.Results:1. Four mAbs against VN were obtained by ELISA screening, named 3D2,4D11,6G5 and 8D9, respectively.2. Two mAbs 6G5 and 8D9 can specifically recognize serum VN, and detected different expression level of VN in serum of patients wtih liver and the normal subjects (P <0.05).3.6G5 and 8D9 mAbs were paired successfully by sandwich ELISA assay and Fortebio system screening.4. When using paired 6G5 and 8D9 to test sera VN of normal individuals and paitents with hepatitis and cirrhosis, dramatic difference (P<0.001) were found between different liver diseases.Conclusion:Four mAbs probing VN were successfully generated, among which 6G5 can pair with 8D9. Furthermore, different expression pattern of VN in various liver diseases were proved through sanwhich ELISA assay with the matched pair of mAbs. The results suggested that our mAbs recognize VN with high specificity, which can be useful tools in clinical development of detection kit for early diagnosis of liver cancer.