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-GPLD65-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 BE/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.