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

AIM: To select the anti-nasopharyngeal carcinoma scFv from a fully human anti-nasopharyngeal carcinoma single-chain phage antibody library, and identify its characteristics; to express and purify the nasopharyngeal carcinoma positive scFv antibody fused to enhanced green fluorescent protein, and observe its binding capacity to CNE2.METHODS: The single-chain phage antibody 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 nasopharyngeal carcinoma cells was confirmed by immunohistochemistry. The recombinant plasmid EGFP-HNSA033 /pET-25b(+) proved by DNA sequencing was transformed into E.coli BL21(DE3), and induced for fusion expression of EGFP-HNSA033 with IPTG The expressed EGFP-HNSA033 was purified and detected with SDS-PAGE. CNE2 was incubated with the fusion expression in vitro, and the binding bioactivity was observed under the fluorescent microscope. Furthermore, we observe its binding capacity in vivo: injecting the EGFP-HNSA033 by caudal vein into nude mice planted with nasopharyngeal carcinoma 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 CNE2 than HUVEC and NP69 and QSG7701 were picked out of 4212 clones. One clone, HNSA033, was further analyzed after DNA sequencing. The results of immunohistochemistry with cultured cells were similar to those of ELISA. HNSA033 reacted specifically to nasopharyngeal carcinoma cells in most human nasopharyngeal carcinoma tissue sections (75%) but in few human normal nasopharyngeal tissue sections (13.3%) . The distinction of positive rates is of a great statistical significance (P<0.05) . E.coli BL21(DE3) containing recombinant plasmid could be observed sending out green fluorescence under fluorescent microscope. The EGFP-HNSA033 protein was expressed in E.coli as inclusion body. The result of immunofluorescent detection verified that scFv HNSA033 could specificially bind to CNE2 cells.CONCLUSION: ELISA, immunohistochemistry and immuno-fluorescence detection results confirm HNSA033 bind specifically with nasopharyngeal carcinoma cells. The scFv fragment against nasopharyngeal carcinoma may be further developed and applied in clinical diagnosis and therapy of nasopharyngeal carcinoma.