Sensitive monoclonal antibody-based sandwich ELISA for determination of the diabetes-associated autoantigen glutamic acid decarboxylase GAD65.
Although various methods for the detection of autoantibodies against glutamic acid decarboxylase (GAD65-AAb) are known, no sensitive method for the quantification of GAD65 as autoantigen is available. We describe a sandwich ELISA based on monoclonal GAD65 antibodies (Mc-GAD65-Ab) of different epitope specificities to quantify GAD65 in pancreatic islets and in different organ/cell extracts and during the preparation of GAD from brain extracts. GAD65 was captured via solid phase coated Mc-GAD65-Ab and detected via a second biotin-labelled Mc-GAD65-Ab recognizing a NH2-terminal epitope of the molecule. The detection limit was estimated to be 0.03 ng GAD65/ml using alkaline phosphatase (AP)-conjugated streptavidin. GAD65 contents in islets of neonatal BB/OK rats and Lewis rats amounted to 37.4 and 43.7 pg/islet, respectively. Furthermore, GAD65 was quantified in brain extracts of pig (55.1 ng/mg protein), mouse (39.5 ng/mg), rat (243.8 ng/mg) and pig cerebellum (514.8 ng/mg) and in different organ extracts of Lewis rat.
Schlosser
M
M
Ernst-Moritz-Arndt-University of Greifswald, Institute of Pathophysiology, Karlsburg, Germany.
Hahmann
J
J
Ziegler
B
B
Augstein
P
P
Ziegler
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001323:Autoantibodies / Q000032:analysis*; D001324:Autoantigens / Q000032:analysis*; D001923:Brain Chemistry; D002460:Cell Line; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018604:Epitope Mapping; D005260:Female; D005968:Glutamate Decarboxylase / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007515:Islets of Langerhans / Q000737:chemistry; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008970:Molecular Weight; D051381:Rats; D011917:Rats, Inbred Lew; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase