Schlosser M, Hahmann J, Ziegler B, Augstein P, Ziegler M
Ernst-Moritz-Arndt-University of Greifswald, Institute of Pathophysiology, Karlsburg, Germany.
J Immunoassay. 1997 Nov;18(4):289-307. doi: 10.1080/01971529708005823.
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.
尽管已知多种检测抗谷氨酸脱羧酶自身抗体（GAD65-AAb）的方法，但尚无用于将GAD65作为自身抗原进行定量的灵敏方法。我们描述了一种基于具有不同表位特异性的单克隆GAD65抗体（Mc-GAD65-Ab）的夹心ELISA，用于定量胰岛、不同器官/细胞提取物以及从脑提取物中制备GAD过程中的GAD65。GAD65通过固相包被的Mc-GAD65-Ab捕获，并通过识别该分子NH2末端表位的第二种生物素标记的Mc-GAD65-Ab进行检测。使用碱性磷酸酶（AP）偶联的链霉亲和素时，检测限估计为0.03 ng GAD65/ml。新生BB/OK大鼠和Lewis大鼠胰岛中的GAD65含量分别为37.4和43.7 pg/胰岛。此外，还对猪（55.1 ng/mg蛋白质）、小鼠（39.5 ng/mg）、大鼠（243.8 ng/mg）和猪小脑（514.8 ng/mg）的脑提取物以及Lewis大鼠的不同器官提取物中的GAD65进行了定量。
