Affinity Maturation Of A Single Chain Variable Fragment Antibody For Hepatocellular Carcinoma

Part 1 Construction and screening of a single-chain Fv antibody mutant library for hepatocellular carcinomaObjective: To select the hepatoma cell-specific antibody from a constructed single-chain Fv antibody mutant library for hepatocellular carcinoma (HCC) by mimicking affinity maturation in vitro.Methods: A single-chain Fv antibody (scFv) mutant library for HCC was established after the heavy chain and light chain of the primitive scFv A4-16 for HCC were mutated by using error-prone PCR and DNA shuffling. Hepatoma cell-specific scFvs were selected after four rounds of panning by phage display and ELISA detection. The specificity for HCC of those selected scFvs was detected by ELISA.Results: The scFv mutant library for HCC was established, the insertion ratio of scFv gene was 91%, so that the real content of the mutant library was 4.5 ×10~7. Four selected positive clones M1 M2 M3 and M4 were obtained after four rounds of panning and ELISA detection. Immunoassay showed that these four positive clones could bind to human HCC cells specifically, but couldn't bind to normal liver cells carcinoma of colon cells gastric cancer cells and carcinoma of pancreas cells.Conclusions: (1) Error-prone PCR combining DNA shuffling was an effective and simple method for affinity maturation of antibodies isolated from phage antibody library. (2) All of the selected positive clones M1 M2 M3 and M4 had high specificity for HCC as the primitive clone A4-16.Part 2 Measurement of relative affinities of HCC phage antibodies and analysis of amino acid sequences of HCC scFvObjective: To compare relative affinities of HCC phage antibodies between the four positive clones and the primitive clone A4-16, and analysis the mutated amino acids in the sequences of the four positive clones, and evaluate the relative affinity of the new mutant...