United Kingdom (UK)
Recombinant monoclonal antibody to CD28. Manufactured using AbAb’s Recombinant Platform with variable regions (i.e. specificity) from the hybridoma 9.3.
UniProt Accession Number of Target Protein: P10747
Alternative Name(s) of Target: T-cell-specific surface glycoprotein CD28; TP44
Immunogen: The original antibody was generated by immunizing mice with human T cells.
Specificity: The antibody is specific for human CD28. CD28 is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. T cell stimulation through CD28 in addition to the T-cell receptor (TCR) can provide a potent signal for the production of various interleukins.
Application Notes: The antibody was found to induce T cell activation and proliferation in collaboration with TPA. Further, T cell proliferation was associated with IL-2 receptor expression and IL-2 production. The antibody stained 60-80% of the isolated T cells by immunofluorescence. The antibody immunoprecipitated CD28 from Jurkat cells. (Hara et al., 1985; PMID: 3159820). Immunofluorescence was performed on T cells expressing CD28 using this antibody. The antibody was cytotoxic for 50-80% of peripheral T cells and for 20-50% of thymocytes but it was not cytotoxic for cultured B-cell lines, normal B cells, or monocytes. In immune precipitation assays, the antibody reacted with CD28 (Hansen et al., 1980). The antibody induces proliferative responses in human T cells in in vitro experiments (Hutloff et al. 1999; PMID: 9930702). The antibody detected CD28 by western blot analysis (Siefken et al,1998; PMID: 9712026). Humanize, chimeric and hybrid Fab versions of the antibody were constructed. The ability of the three Fabs to bind to CD28 was examined by ELISA; the three Fabs showed a binding activity, with an EC50 of 630, 20 and 30 nM respectively for the humanized, the chimeric, hybrid Fab version. Thus, the humanized Fab showed a moderate loss in avidity. The three Fab versions of the antibody were tested for blocking ligation of CD28-Ig to CHO cells expressing CD80 and the binding was quantified by flow cytometry. The chimeric Fab exhibited the best blocking activity followed by the hybrid and the humanized Fab, with EC50 values of 0.2, 0.4, and 1.2 µM, respectively. Lastly, three Fab versions showed biological activity as an immunosuppressant by inhibiting a MLR (Tan et al. 2002; PMID: 12097421).
Antibody first published in:
Hansen et al. Monoclonal antibodies identifying a novel T-Cell antigen and Ia antigens of human lymphocytes Immunogenetics. 1980; 10:247. PMID:
Note on publication:
The original paper describes the generation and characterization of the antibody.
Flow cytometry using anti-CD28 antibody 9.3 (Ab03228). Jurkat cells were stained with the anti-unknown specificity antibody (Ab00178-23.0; isotype control, black line) or the rabbit IgG version of 9.3 (Ab03228-23.0, blue line) at a dilution of 1:100 for 1h at RT. After washing, the bound antibody was detected using a goat anti-rabbit IgG AlexaFluor® 488 antibody at a dilution of 1:1000, and the cells were analyzed using a FACSCanto flow-cytometer.
Immunofluorescence staining of Jurkat cells with anti-CD28 antibody 9.3 (Ab03228). Immunofluorescence analysis of paraformaldehyde fixed Jurkat cells on Shi-fix™ coverslips stained with the chimeric rabbit IgG version of 9.3 (Ab03228-23.0) (1:100 dilution) for 1h followed by Alexa Fluor® 488 secondary antibody (1:1000 dilution), showing membrane and cytoplasmic staining. The nuclear stain is DAPI (blue). Panels show, from left-right, top-bottom, Ab03228-23.0, DAPI, merged channels, and an isotype control. The isotype control was an unknown specificity antibody (Ab00178-23.0) followed by staining with Alexa Fluor® 488 secondary antibody.