Waldrop Megan A, Suckow Arthur T, Hall Tyler R, Hampe Chris S, Marcovina Santica M, Chessler Steven D
Department of Medicine, University of California, San Diego, La Jolla, California 92093-0726, USA.
Diabetes Technol Ther. 2006 Apr;8(2):207-18. doi: 10.1089/dia.2006.8.207.
Glutamic acid decarboxylase-65 (GAD65) is a major autoantigen in autoimmune diabetes and is discharged from injured islet beta cells. GAD65 may also be released by transplanted islets undergoing immunological rejection. To test hypotheses regarding the utility of GAD65 as a biomarker for transplant rejection or diabetes-associated islet damage and also regarding the timing and instigators of GAD65 release in humans or animal models, a sensitive assay capable of measuring GAD65 in serum or plasma will be necessary. Ideally, this assay would also be resistant to interference by anti-GAD65 autoantibodies.
A novel, magnetic bead-based assay was developed based on GAD65 capture by a monoclonal antibody directed to the only region of the protein known not to be significantly targeted by autoantibodies. A subsequent denaturation step allows sensitive immunodetection to proceed using anti-GAD65 polyclonal antibodies that would otherwise potentially be blocked by bound autoantibodies.
The GAD65 assay worked equally well with serum and plasma as with a solution of bovine serum albumin (BSA). The limit of blank was 31 pg/mL and did not differ significantly in the BSA solution (27 pg/mL). Mean recovery of GAD65 from the plasma of control subjects and GAD65 autoantibody-positive and -negative subjects with type 1 diabetes was 101 +/- 4.6%, 88 +/- 7.8%, and 99 +/- 7.0% (+/- SEM), respectively. The assay was used to quantify both recombinant GAD65 and the GAD65 content of human and rodent islets and other tissue extracts that were added to human plasma samples.
A sensitive, autoantibody-resistant GAD65 assay has been developed that is compatible with detection in serum and plasma and therefore will likely also work with a variety of other biologic fluids. This assay may enable the use of circulating GAD65 as a biomarker of islet damage or transplant rejection and will facilitate in vivo studies of the pathogenesis of anti-GAD65 autoreactivity.
谷氨酸脱羧酶65（GAD65）是自身免疫性糖尿病中的主要自身抗原，由受损的胰岛β细胞释放。GAD65也可能由经历免疫排斥的移植胰岛释放。为了验证关于GAD65作为移植排斥或糖尿病相关胰岛损伤生物标志物的效用，以及关于GAD65在人类或动物模型中释放的时间和诱因的假设，需要一种能够测量血清或血浆中GAD65的灵敏检测方法。理想情况下，这种检测方法还应能抵抗抗GAD65自身抗体的干扰。
基于一种单克隆抗体捕获GAD65开发了一种新型的基于磁珠的检测方法，该单克隆抗体针对该蛋白质唯一已知未被自身抗体显著靶向的区域。随后的变性步骤允许使用抗GAD65多克隆抗体进行灵敏的免疫检测，否则这些多克隆抗体可能会被结合的自身抗体阻断。
GAD65检测在血清、血浆以及牛血清白蛋白（BSA）溶液中表现同样良好。空白限为31 pg/mL，在BSA溶液中（27 pg/mL）无显著差异。1型糖尿病对照受试者、GAD65自身抗体阳性和阴性受试者血浆中GAD65的平均回收率分别为101±4.6%、88±7.8%和99±7.0%（±SEM）。该检测方法用于定量重组GAD65以及添加到人类血浆样本中的人和啮齿动物胰岛及其他组织提取物中的GAD65含量。
已开发出一种灵敏的、抗自身抗体的GAD65检测方法，该方法与血清和血浆检测兼容，因此可能也适用于多种其他生物体液。这种检测方法可能使循环GAD65能够用作胰岛损伤或移植排斥的生物标志物，并将有助于抗GAD65自身反应性发病机制的体内研究。
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