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 cytoxicity (C'AMC) to rat islet cells. Whereas our monoclonal Beta-cell specific surface antibody K14D10 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.
Ziegler
B
B
Institut für Diabetes Gerhard Katsch Karlsburg, Ernst-Moritz-Arndt Universität Greifswald, Germany.
Augstein
P
P
Schröder
D
D
Mauch
L
L
Hahmann
J
J
Schlosser
M
M
Ziegler
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002860:Chromium Radioisotopes; D003165:Complement System Proteins / Q000276:immunology; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005968:Glutamate Decarboxylase / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007074:Immunoglobulin G / Q000276:immunology; D007515:Islets of Langerhans / Q000166:cytology / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats
D000911:Antibodies, Monoclonal; D002860:Chromium Radioisotopes; D007074:Immunoglobulin G; D008562:Membrane Glycoproteins; D003165:Complement System Proteins; D005968:Glutamate Decarboxylase