Development of novel diabetes autoantibody assays based on luciferase reporters

描述(申请人提供)：1A型糖尿病(T1D)的特征是由于胰岛β细胞的自身免疫破坏而导致的绝对胰岛素不足，是困扰美国儿童的最常见的自身免疫性内分泌疾病。它承担着巨大的社会和卫生保健成本，并以每年约2%-5%的速度增长。糖尿病自身抗体是目前最有力的疾病生物标志物，对诊断和基因风险分层至关重要。胰岛素自身抗体(IAA)通常是最先出现的，尤其是在幼儿中，它们的测量可以识别哪些人最有可能从预防性胰岛素治疗中受益。这使得IAA的可靠检测变得尤为重要。然而，目前使用的检测方法在标准化测试中表现不佳，显然需要改进。我们正在进行的针对锌转运蛋白8(ZNT8A)自身抗体的研究表明，通过对用于检测它们的探针进行修改，可以显著提高检测的特异性和灵敏度。我们的假设是，我们可以应用从优化ZNT8A检测方法中获得的经验来开发一种改进的检测IAA的程序，该程序基于新型含荧光素酶的探针的使用。我们的初步数据表明，使用人胰岛素原(HPPI)和修饰的赤霉菌荧光素酶(GLuc)之间的融合蛋白可以检测到IAAs，这种融合蛋白具有更高的发光稳定性。当前建议的单一特定目标是优化这种基于hPPI-GLuc的IAA检测。首先，这将涉及对该探针的关键特征的系统研究，例如包含多个结构域、C肽的切除潜力以及胰岛素成分(人和猪)的序列。随后，我们将优化探针的生产和检测程序的基本特征，如糖尿病血清和探针之间的孵育时间和温度，以及收集产生的免疫复合物的方法。我们认为，目前的建议将导致开发一种IAA分析方法，其特异性和灵敏度至少与目前使用的MIAA方法相当，而且不涉及使用放射性同位素，具有更大的效用。基本的探针设计也可以被修改以加入其他蛋白质抗原，因此我们的建议可能对诊断除T1D之外的各种其他自身免疫性疾病具有重要的实用价值。 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，在以后的生活中有很高的破坏性并发症的风险。自身抗体是疾病最强大的生物标志物，我们的建议将开发改进的程序来测量胰岛素自身抗体，目前显示的重复性很差。胰岛素是1A型糖尿病的主要分子靶点之一，尤其是在幼儿中，我们的建议将有助于诊断，并有助于确定哪些人可能从预防性胰岛素治疗中受益最大。

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.