Analysis of the human anti TNF α Fab obtained with epitope guided selection
Objective To characterize the clones of human anti TNF α Fab fragments obtained with epitope guided selection. Methods The variable regions of antibodies were sequenced with competitive ELISA to determine whether the humanized antibody Fabs and the parental monoclonal antibody Z8 could recognize the same epitope. The in vitro neutralization assay was used to test the biological activity of humanized antibody Fabs. The relative affinity of the humanized antibody Fabs were determined with ELISA using thiocyanate elution. Results Sequencing showed that the heavy variable regions of two antibody Fabs were same, which belonged to V H Ⅰsubgroup, and the light variable regions belonged to VκⅠand VκⅢ, respectively. Functional analysis demonstrated that human Fab resembled its parental mouse McAb Z8 in that they both recognized the same epitope and neutralized the cytotoxicity of TNF α. The affinity of humanized Fabs were lower than that of Z8. Conclusions The human Fabs obtained with epitope guided selection had similar characteristics of their parental mouse McAb.