Glutamate Decarboxylase (GAD) is not Detectable on the Surface of Rat Islet Cells Examined by Cytofluorometry and Complement-Dependent Antibody-Mediated Cytotoxicity of Monoclonal GAD Antibodies

After fusion of splenocytes from a Balb/c mouse which received three injections of human recombinant GAD₆₅ 44 hybridomas producing monoclonal GAD antibodies (mc-GAD Ab) could be established. 35 out of the 44 mc-GAD Ab specifically recognized the GAD₆₅ isoform, whereas 9 showed a cross-reactivity with GAD₆₇. All antibodies belong to the IgG class. The mc-GAD Ab were reactive with recombinant as well as natural GAD tested in enzyme-linked immunosorbent assay. Twenty-one antibodies stained islet cells very well in cryosections of human, monkey, rat, and pig pancreas. However, the mc-GAD Ab failed to bind on the surface of viable rat islet cells, although they reveal a striking binding on permeabilized cells examined by cytofluorometry. Furthermore, the mc-GAD Ab were analyzed for complement-dependent antibody-mediated cytotoxicity (C'AMC) to rat islet cells. Whereas our monoclonal Beta-cell specific surface antibody K14D1O caused a high C'AMC measured as a ⁵¹Cr-release of 56.5 ± 4.6%, n = 10, only 4/44 mc-GAD Ab provoked a moderate increase of ⁵¹Cr-release ranging from 7.1-10.5% (cut-off 7.0%). From these findings it is suggested that GAD is not detectable on the islet-cell surface by binding and cytotoxicity test.

Keywords

Monoclonal antibodies - Glutamic acid decarboxylase isoforms - Rat islet cells - Immunostaining - Complement-dependent antibody-mediated cytotoxicity