Serological Identification Of Phage Antibody Of Single Chain Variable Fragment Of Humam Hepatocellular Cell Carcinoma

Objective: To study whether the specific scFvs-scFv4-16 against hepatocellular carcinoma which our laboratory had been constructed and screened would have specificity for blood serum of human heptocellular carcinoma, and to evaluate the serological diagnostic value of scFv4-16 in patients with HCC.Method: 1. The scFv4-16 would be cloned from E.coli TGI and infected E.coli HB2151. After induced by IPTG, the soluble scFvs4-16 were expressed in the bacteria periplasm of E.coli HB2151 abundantly. Using HiTrap Anti-E Tag column, the scFvs4-16 were purified by affinity chromatography. SDS-polyacrylamide gel electrophoresis of purified scFvs was analyzed. The consistencies and bioactivity of purified scFvs4-16 were measured by Bradford method. Then the expressed scFv4-16 would be concentrated and conserved.2. Preliminary tests were performed to found the optimal ELISA condition that include the reaction time and the concentration of the scFv4-16, enzyme-labelled secondary antibody, coated serum. Establish the optimization of indirect ELISA condition.3. The optical density value was detected by convenient indirect ELISA whether the scFvs4-16 had responded to the blood serum of human hepatocellular carcinoma, and the blood serum of health adult, the digestive system cancer , nontumorous patients of chronic liver disease were used as control. The rates of positive responses were calculated and compared with AFP. 4.Moreover, the rates of the positive response of blood serum of HCC to scFv4-16 were compared with that of AFP. 5.To study the clinical value of combined detection AFP andscFv4-16 in the diagnosis of hepatocellular carcinoma.Result: 1. The purified scFv4-16 against hepatocellular carcinoma was secreted into the periplasm of host bacterisa abundantly. SDS-polyacrylamide gel electrophoresis analysis of purified scFvs showed that a single protein band about 31 KD.Then the expressed scFv4-16 was concentrated and conserved.. The yield of purified protein was 20μg/ml. 2. The indirect ELISA is stable, sensitive and specific comparatively. 3. There were significantly differences in OD values of HCC patient's serum compared with that of health adult, the other digestive system cancer,...