Characterization of Novel Fragment Antibodies Against TNF-alpha Isolated Using Phage Display Technique

Tumor necrosis factor alpha (tnf-α) is an inflammatory cytokine which plays crucial roles in pathogenesis of inflammatory diseases. the current study aimed to investigate the binding abilities of i44 and i49 domain antibodies to tnf-α. the dabs were expressed in bacterial expression system and purified by affinity chromatography using ni-sepharose column. the expression and purity of the proteins were evaluated using western blotting and sds-page techniques, respectively. elisa experiment showed that i44 and i49 dabs bind to tnf-α with the binding constants (kd) of 5.18 ± 1.41 and 2.42 ± 0.55 μm, respectively. the inhibitory effect of dabs on tnf-α biological effect was determined in mtt assay in which i44 and i49 prevented tnf-α cell cytotoxicity with ic_50 values of 6.61 and 3.64 μm, respectively. the identified anti-tnf-α dabs could bind to and inhibit tnf-α activity. the dabs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with tnf-α. the results of the current study can pave the way for further structural studies in order to introduce new more potent anti-tnf-α antibodies.