Preparation of functional monoclonal antibody against human CD28 and analysis of its biological feature

Aim To prepare the monoclonal antibodies (mAbs) against human CD28 and to study its biological feature. Methods The hybridoma cell lines were obtained by fusing spleen cells of Blab/c mice that had been immunized with murine lymphoma cells transfected with full-length huaman CD28 cDNA to myeloma cells Sp2/0. Ascites were induced to produce the mAbs. The specificity and affinity of the mAb 18G8 was verified by CD28 competitive inhibitory test and FACS. Reactivities of mAb 18G8 to PBTC, U266, 8226, Jurkat and Daudi cell were studied by indirect immunofluorescence staining. mAb 18G8-inducing proliferation of peripheral blood T cells (PBTCs) was determined by [3H]thymidine incorporation test. Results Five hybridoma cell lines were obtained. mAb 18G8 secreted by one of the them, belong to mouse IgG2a. It recognized a epitope different from which recognized by the standard mAb(clone CD28.2). The Reactivitrates of the mAb 18G8 to PBTC, U266, 8266, Jurkat and Daudi cells were 70.2％, 99.3％, 98.6％, 76.4％and 1.9％, respectively, similar with CD28.2. It was indicated that different antigen epitopes expressed on all above cells. mAb 18G8 could promote the PBTC proliferation in vitro(SI=7). It was indicated that The substitution of mAb 18G8 for B7-1 molecule could also mediate the costimulatory signals. Conclusion 18G8 is a specific and functional anti-CD28 mAb it may be of significant value in basic studies and clinical application.