Development of novel diabetes autoantibody assays based on luciferase reporters

描述（由申请人提供）：1A型糖尿病（T1 D）的特征是胰腺β细胞的自身免疫性破坏导致的绝对胰岛素不足，是美国儿童最常见的自身免疫性内分泌疾病。它承担着巨大的社会和医疗保健成本，并以每年约2 - 5%的速度增长。糖尿病自身抗体是目前最强大的疾病生物标志物，是诊断的核心，并对那些遗传风险分层。胰岛素自身抗体（IAA）通常是最先产生的，特别是在幼儿中，它们的测量可以识别最有可能从预防性胰岛素治疗中受益的个体。这使得IAAs的可靠测定特别重要。然而，目前使用的测定在标准化测试中表现不佳，并且显然需要改进。 我们正在进行的针对锌转运蛋白8（ZNT 8A）的自身抗体的研究已经证明，可以通过对用于其检测的探针进行修改来实现对测定特异性和灵敏度的显著改善。我们的假设是，我们可以应用我们从优化ZNT 8A测定中获得的经验来开发基于使用含有新型荧光素酶的探针的用于测量IAAs的改进程序。我们的初步数据表明，IAAs可以使用人前胰岛素原（hPPI）和改良的高斯princeps荧光素酶（GLuc），具有增强的发光稳定性之间的融合蛋白进行检测。当前提议的单一具体目标是优化这种基于hPPI-GLuc的IAA测定。最初，这将涉及对探针关键特征的系统研究，例如包含多个结构域、切除C肽的可能性和胰岛素组分的序列（人v猪）。随后，我们将优化探针生产和基本特征的测定程序，如糖尿病血清和探针之间的孵育时间和温度，以及所得免疫复合物的收集方法。 我们认为，目前的建议将导致开发一种测定IAAs的方法，该方法至少具有与目前使用的mIAA相当的特异性和灵敏度，并且具有更大的实用性，不涉及使用放射性同位素。基本的探针设计也可以适用于纳入其他蛋白质抗原，因此我们的建议可以有显着的效用，除了T1 D的各种其他自身免疫性疾病的诊断。 公共卫生相关性：1A型胰岛素依赖型糖尿病是儿童和年轻人最常见的慢性疾病之一，并且在以后的生活中具有毁灭性并发症的高风险。自身抗体是疾病的最强大的生物标志物，我们的建议将开发用于测量胰岛素自身抗体的改进程序，目前显示重现性差。胰岛素是1A型糖尿病的主要分子靶点之一，特别是在幼儿中，我们的建议将有助于诊断，并确定可能从预防性胰岛素治疗中获益最多的个体。

DESCRIPTION (provided by applicant): Type 1A diabetes (T1D) is characterized by an absolute insulin insufficiency resulting from autoimmune destruction of pancreatic beta cells, and is the most common autoimmune endocrine disease afflicting children in the US. It is responsible for significant societal and health care costs, and is increasing at a rate of approximately 2 - 5% per year. Diabetes autoantibodies are currently the most robust biomarkers of disease, and are central to diagnosis, and for stratification of those genetically at risk. Autoantibodies to insulin (IAA) are typically the first to develop, especially in young children, and their measurement may enable identification of individuals who are most likely to benefit from prophylactic insulin therapy. This makes a reliable assay for IAAs particularly important. However the presently used assay performs poorly in standardization tests, and is clearly in need of improvement. Our ongoing studies of autoantibodies to zinc transporter 8 (ZNT8A) have demonstrated that significant improvements to assay specificity and sensitivity can be achieved by making modifications to the probe used for their detection, Our hypothesis is that we can apply the experience we have gained from optimizing the ZNT8A assay to develop an improved procedure for measuring IAAs, based upon the use of novel luciferase containing probes. Our preliminary data indicate that IAAs can be detected using a fusion protein between human preproinsulin (hPPI) and a modified Gaussia princeps luciferase (GLuc) that has enhanced light emission stability. The single specific aim of the current proposal is to optimize this hPPI- GLuc based IAA assay. Initially this will involve a systematic investigation of key features of the probe, such as inclusion of multiple domains, potential for excision of the C peptide, and sequence of the insulin component (human v porcine). Subsequently we will optimize probe production and basic features of the assay procedure such as length and temperature of the incubation between diabetic sera and the probe, and the method of collection of the resulting immune complexes. We believe that the current proposal will lead to the development of an assay for IAAs that has at least equivalent specificity and sensitivity to the currently used mIAA, with the greater utility of not involving the use of radio-isotopes. The basic probe design can also be adapted to incorporate other protein antigens, and hence our proposal could have significant utility for the diagnosis of a variety of other autoimmune diseases, in addition to T1D. PUBLIC HEALTH RELEVANCE: Type 1A insulin dependent diabetes mellitus is one of the most frequent chronic diseases of children and young adults, and carries a high risk of devastating complications in later life. Autoantibodies are the most robust biomarkers of disease, and our proposal will develop improved procedures for measuring autoantibodies to insulin, which currently show poor reproducibility. Insulin is one of the major molecular targets in type 1A diabetes, especially in young children, and our proposal will facilitate diagnosis, and the identification of individuals who may benefit the most from preventative insulin therapy.