Investigation of the production, purification and binding ability of anti-TNF-α scFv J48 antibody identified by phage display technique.

Abstract

Introduction: TNFα is one of the cytokines which is responsible for inflammatory responses. In this context, using antibodies with smaller size and stronger binding affinity can be considered as a promising strategy to inhibit TNFα activity in inflammatory disease. Aims: In the current investigation, the ScFv J48 antibody was produced and purified against TNFα. Binding ability and inhibitory effect of J48 to TNFα was examined by ELISA and MTT assay. The possible interactions among TNFα and J48 was investigated by docking studies. Methods: The stop codon of the J48 coding sequence, was turned to the tyrosine codon with site directed mutagenesis. Then the generated sequence was cloned into the pET28a. The vector containing the gene encoding J48 was transformed into E. coli Bl21 plysS. The produced antibody was purified by affinity chromatography columns. Protein expression was checked by SDS-PAGE and western blotting techniques. ELISA experiment was used to examine binding ability of J48 to TNFα. MTT assay was carried out to assess J48 ihibitory on TNFα cytotoxicity. Docking studies were conducted to estimate the possible interactions between J48 and TNFα. Results: The J48 antibody was expressed in prokaryotic cells. The result of SDS-PAGE showed a protein bound around 25kDa. Western blotting analysis confirmed the production of J48 antibody. ELISA results showed that J48 has a relatively high binding affinity to TNFα. MTT assay confirmed that J48 could inhibit TNFα cytotoxicity. Docking results revealed that the hydrogen bond, hydrophobic contents were involved in the interactions between TNFα and J48. Conclusions: In this work, J48 antibody was expressed in bacterial expression system. According to the ELISA and MTT results, J48 has a good binding and inhibition activity on TNFα. J48 can be a suitable candidate for in-vivo studies and also be used for designing anti-TNFα medications.