Selection And Identification Of A Fully Humanized Anti-hepatoma Single-chain Fv Fusion Phage Antibody And Its Bioactivity Detection In Vivo

AIM: To select on a large scale and identify the anti-hepatoma scFv from scFv-displaying phage library; to express and purify the hepatoma positive scFv antibody fused to enhanced green fluorecsent protein, and observe its binding capacity to HepG₂.METHODS: A fully human scFv-displaying phage library was subjected to three rounds of positive and negative cell panning and enrichment and then it was selected by ELISA. The binding specificity of phage antibodies with hepatoma carcinoma cells was confirmed by immunohistochemistry. The recombinant plasmid EGFP-SA3 /pET-25b(+) proved by DNA sequencing was transformed into E.coli BL21(DE3), and induced for fusion expression of EGFP-SA3 with IPTG. The green fluorescence of E.coli BL21(DE3) harboring recombinant plasmid was observed under the fluorecsent microscope. The expressed EGFP-SA3 was purified and detected with SDS-PAGE. HepG₂ was incubated with the recombinant EGFP-SA3 in vitro, and the binding bioactivity was observed under the fluorecsent microscope. Further more, we observe its binding capacity in vivo: injecting the EGFP-SA3 by caudal vein into nude mice planted by hepatoma and observing the whole body fluorescence image of EGFP.RESULTS: After panning, enrichment and testing by ELISA, 3 phage antibody clones reacting more strongly to HepG₂ than QSG-7701 were picked out of 2798 clones. One clone, SA3, was further analysed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. SA3 reacted specifically to hepatoma cells in most human hepatoma tissue sections (72.1 %) but in few human normal liver tissue sections (8.3%) . The distinction of positive rates is of a great statistical significance (P<0.01) . E.coli BL21(DE3) containing recombinant plasmid could be observed sending out green fluorescence under fluorecsent microscope. The EGFP-SA3 protein was expressed in E.coli as inclusion body. The result of immunofluorecsent detection verified that scFv SA3 could specificially bind to HepG₂ cells.CONCLUSION: ELISA, immunohistochemistry and immuno-fluorescence detection results conform SA3 bind specifically with hepatoma carcinoma cells. The scFv fragment against hepatoma may be further developed and applied in clinical diagnosis and therapy of liver cancer.