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

ABSTRACT: Tumor necrosis factor alpha (TNF-?) 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-?. 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-? with the binding constants (Kd) of 5.18 ± 1.41 and 2.42 ± 0.55 µM, respectively. The inhibitory effect of dAbs on TNF-? biological effect was determined in MTT assay in which I44 and I49 prevented TNF-? cell cytotoxicity with IC50 values of 6.61 and 3.64 µM, respectively. The identified anti-TNF-? dAbs could bind to and inhibit TNF-? activity. The dAbs activities can be attributed to their ability to establish hydrogen bonds as well as hydrophobic contacts with TNF-?. The results of the current study can pave the way for further structural studies in order to introduce new more potent anti-TNF-? antibodies.

SUBMITTER: Alizadeha AA 

PROVIDER: S-EPMC6706722 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature