Application of Indirect ELISA and PCR Techniques for Detecting of Hepatocellular Carcinoma using Des-gamma Carboxyprothrombin, Alpha-fetoprotein, and Thioredoxin Biomarkers.

Abstract

Hepatocellular carcinoma (HCC) is one of the five most common cancers and the second leading cause of cancer-related death worldwide. In this study, three monoclonal antibodies were developed for the early detection of HCC. The enzyme-linked immunosorbent assay (ELISA) method is used to detect antigens causing HCC. The final working dilutions of the coated antigen, monoclonal antibody, and enzyme-labeled secondary antibody were determined to be 1:5, 1:100, and 1:15,000, respectively. The optimal dilution of blocking buffer was 1.5% BSA phosphate buffer. The cutoff values were determined to be 0.1989, 0.2539, and 0.3059 for the Des-gamma carboxyprothrombin (DCP), Alpha-fetoprotein (AFP) and Thioredoxin (TXN) antigens, respectively. There is no cross-reaction between antigens and antibodies of different types. The coincidence rates between the indirect ELISA and commercial kits for detecting DCP, AFP, and TXN antigens were 95.24%, 95.24%, and 96.83%, respectively. In addition, a procedure to detect genes encoding TXN, DCP, and AFP via PCR has been developed. The results of the indirect ELISA and PCR methods are similar. In summary, we successfully constructed an indirect ELISA method to detect HCC-causing antigens via three monoclonal antibodies and designed primers to amplify HCC-causing gene fragments, which can be used for diagnosis and screening in clinical medicine.