Isolation of anti-Hsp70 antibody fragment from phage antibody library

Abstract

Hsp70 is a heat shock protein that is either not expressed in normal cells or has low level expression whereas it has three forms including cytoplasmic, membrane and extracellular in various cancers and participates in different stages of tumorigenesis. Purpose: production, purify and characterization of an anti-Hsp70 single-chain variable fragment antibody. Material and Methods: By studying the three-dimensional structure of Hsp70, a 12 amino acid region was identified as a target. This peptide was chemically synthesized and used to screen the phage library of human antibody fragments. After four rounds of biopanning, the specificity of the obtained clones was determined using polyclonal and monoclonal phage ELISA. The accuracy of the DNA sequences of the positive colonies was evaluated by PCR and sequencing. After expression of the selected clone in HB2151 host and purification by affinity chromatography, performance of the antibody fragment was evaluated by dot blotting and immunofluorescence microscopy, then its binding kinetics to the target peptide was quantitatively quantified by SPR technique. Results: According to PCR results, 8 colonies out of 14 colonies had both VH and VL genes. But DNA sequence analysis showed that only 5 colonies had acceptable sequences. The selected clone (G6A) had to express about 17.4 kDa antibody fragment due to its shorter VH gene. Both expression of G6A antibody fragment within the expected weight range and its high purity after affinity chromatography were demonstrated by SDS-PAGE and western blotting. Specificity, function and kinetics of binding were also confirmed by dot blot, immunofluorescence microscopy and SPR. Conclusion: In this study, a full human anti-Hsp70 antibody fragment was produced, purified and proven to be a suitable platform for the development of therapeutic and diagnostic agents against Hsp70 expressing cancers.