Screening and preliminary identification of human single chain variable fragments antibAies against medullary thyroid cancer

Abstract:

Objective：To isolate single chain variable fragments（scFv） antibAies against medullary thyroid carcinoma（MTC） from a large human phage display library and to identify the biological activity of positive clones. Methods：Thyroid epithelial cell line（TEC cell） and MTC cell line（TT cell） were used as negative and positive antigen respectively and then five rounds of ‘adsorption elution-amplification’from the large human phage display library against MTC was conducted. Specific antibAy was tested by ELISA. E.coli HB2151 was infected to induce the expression of soluble scFv. Soluble scFv was purified by HiTrapTM Anti E-tag column. The ex－pression and relative molecular weight of the soluble scFv were detected by SDS-PAGE and Western blot. Immune activity of specific soluble scFv was detected by cell ELISA. They were divided into four groups：TT cells group，SW 480 cells group，TEC cells group and control group with PBS；A450 nm was detected by microplate reader respectively. The effects of proliferation of TT cell at different concen－trations were explored by methyl thiazolyl tetrazolium（MTT） assay. They were divided into：TT cells group，SW 480 cells group and control group with PBS，A450 nm absorbance was detected by microplate reader and inhibition rates were calculated. Results：After five panning，antibAies obtained obvious enrichment. Phage ELISA and scFv ELISA results showed that positive reactions to TT cells were 65% and 50% respectively. SDS-PAGE and Western blot showed that the relative molecular weight of the soluble scFv was about 28 kD. Cell ELISA results showed that the absorbance of TT cell group（0.41±0.12） was significantly increased compared with that of SW 480 cells group（0.20±0.03），TEC cell group（0.13±0.01），PBS control group（0.07±0.01）（P= 0.000）. MTT assay showed that when the concentration of scFv was 0.1，1，10 μmol/L，the inhibition rate between the two cell lines was statis－tically significant different（t=2.881，P=0.02；t=5.073，P=0.006；t=7.324，P=0.000）. When the concentration of scFv was 10 μmol/L，the inhibition rate of TT cells reached maximum（0.59±0.15）%. Conclusion：Human anti-MTC is successfully obtained with large phage antibAy library technique.