Oak Shilpa, Radtke Jared, Landin-Olsson Mona, Törn Carina, Lernmark Ake, Hampe Christiane S
Department of Medicine, University of Washington, Building SLU II, 815 Mercer Street, Seattle, WA 98019, USA.
J Immunol Methods. 2009 Dec 31;351(1-2):55-61. doi: 10.1016/j.jim.2009.09.004. Epub 2009 Sep 23.
Anti-idiotypic antibodies (anti-Id) to autoantibodies are present in several autoimmune diseases and are hypothesized to have regulatory function. Recently we reported the presence of anti-Id to a major type 1 diabetes-associated autoantibody (GAD65Ab) in sera of healthy individuals. Our current assay for the detection of GAD65Ab-specific anti-Id requires the initial removal of anti-Id from the sera using immobilized monoclonal GAD65Ab, followed by detection of the now exposed GAD65Ab. However, anti-Id in samples that are GAD65Ab-negative cannot be detected in this assay. Furthermore, we cannot distinguish between serum GAD65Ab and the monoclonal GAD65Ab used in the absorption of anti-Id. In this study we evaluated two novel detection assays for GAD65Ab-specific anti-Id. The biotin/streptavidin based absorption assay utilizes the strong interaction of biotin and streptavidin to prevent possible leakage of the immobilized antibody. Moreover, this assay format allows to identify the origin of the detected GAD65Ab. The ECL-based assay allows the direct detection of anti-Id independent of the presence of GAD65Ab. We analyzed new-onset type 1 diabetes patients (n=133) and matched healthy controls (n=178) for the presence of GAD65Ab-specific anti-Id using both new detection assays and the original absorption assay. We found that all three assays can distinguish between the type 1 diabetes cohort and the healthy control samples. The biotin/streptavidin assay allowed us to positively exclude the monoclonal GAD65Ab as the source of the detected GAD65Ab. While the original absorption assay showed the highest sensitivity and specificity, the ECL format showed the highest peak signal-to-noise ratio and excellent linear correlation, making this assay our first choice for quantification of anti-Id.
针对自身抗体的抗独特型抗体（抗Id）存在于多种自身免疫性疾病中，据推测具有调节功能。最近我们报道了健康个体血清中存在针对一种主要的1型糖尿病相关自身抗体（GAD65Ab）的抗Id。我们目前检测GAD65Ab特异性抗Id的方法需要先用固定化单克隆GAD65Ab从血清中初步去除抗Id，然后检测此时暴露的GAD65Ab。然而，在该检测中无法检测到GAD65Ab阴性样本中的抗Id。此外，我们无法区分血清中的GAD65Ab和用于吸收抗Id的单克隆GAD65Ab。在本研究中，我们评估了两种检测GAD65Ab特异性抗Id的新方法。基于生物素/链霉亲和素的吸收法利用生物素和链霉亲和素之间的强相互作用来防止固定化抗体的可能泄漏。此外，这种检测形式能够确定检测到的GAD65Ab的来源。基于电化学发光（ECL）的检测法可直接检测抗Id，而无需考虑GAD65Ab的存在。我们使用这两种新检测方法和原始吸收法分析了新发病的1型糖尿病患者（n = 133）和匹配的健康对照（n = 178）中GAD65Ab特异性抗Id的存在情况。我们发现所有三种检测方法都能区分1型糖尿病队列和健康对照样本。生物素/链霉亲和素检测法使我们能够肯定地排除单克隆GAD65Ab作为检测到的GAD65Ab的来源。虽然原始吸收法显示出最高的灵敏度和特异性，但ECL形式显示出最高的峰值信噪比和出色的线性相关性，使其成为我们对抗Id进行定量的首选检测方法。
