Development of novel diabetes autoantibody assays based on luciferase reporters

基于荧光素酶报告基因的新型糖尿病自身抗体测定的开发

基本信息

批准号：
8075000
负责人：
HOWARD W DAVIDSON
金额：
$ 7.57万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-06-01 至 2012-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8075000
关键词：
Antibodies Antigen-Antibody Complex Antigens Autoantibodies Autoantigens Autoimmune Diseases Autoimmune Process Beta Cell Biological Assay Biological Markers C-Peptide Cellular Structures Child Chimeric Proteins Chronic Disease Collection Complementary DNA Coupled Data Detection Development Diabetes Mellitus Diabetes autoantibodies Diagnosis Disease Elderly Endocrine System Diseases Excision Family suidae Genetic Transcription Health Care Costs Human In Vitro Individual Insulin Insulin-Dependent Diabetes Mellitus Intervention Investigation Iodination reaction Isotopes Laboratories Lead Length Longitudinal Studies Luciferases Measurement Measures Methods Modification Molecular Target Procedures Production Proinsulin Proteins Protocols documentation Radio Radiolabeled Reaction Reagent Recombinant Proteins Reporter Reproducibility Risk Screening procedure Sensitivity and Specificity Serum Standardization Stratification Structure of beta Cell of islet Temperature Testing Therapy Clinical Trials Transfection Translations assay development base design diabetic experience high risk high throughput screening improved light emission novel preproinsulin programs prophylactic protein aminoacid sequence public health relevance radiotracer tissue/cell culture young adult zinc-binding protein

项目摘要

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.

描述（由申请人提供）：1A 型糖尿病 (T1D) 的特点是胰腺 β 细胞自身免疫性破坏导致胰岛素绝对不足，是美国儿童最常见的自身免疫性内分泌疾病。它造成了巨大的社会和医疗保健成本，并且以每年约 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，对于诊断和对遗传风险人群进行分层至关重要。胰岛素自身抗体（IAA）通常是最先产生的，尤其是在幼儿中，其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 IAA 的可靠测定变得尤为重要。然而，目前使用的测定在标准化测试中表现不佳，显然需要改进。我们正在进行的锌转运蛋白 8 (ZNT8A) 自身抗体研究表明，通过修改用于检测的探针，可以显着提高检测特异性和灵敏度。我们的假设是，我们可以应用从优化中获得的经验ZNT8A 测定法基于使用含有新型荧光素酶的探针，开发了一种改进的 IAA 测量程序。我们的初步数据表明，可以使用人前胰岛素原 (hPPI) 和改良的高斯王子荧光素酶 (GLuc) 之间的融合蛋白来检测 IAA，该融合蛋白增强了光发射稳定性。当前提案的唯一具体目标是优化基于 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、C 肽切除的可能性以及胰岛素成分的序列（人与猪）。随后，我们将优化探针的生产和测定程序的基本特征，例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。我们相信，当前的提案将导致开发一种 IAA 测定方法，该方法至少与目前使用的 mIAA 具有同等的特异性和灵敏度，并且不涉及使用放射性同位素，具有更大的效用。基本探针设计还可以适应其他蛋白质抗原，因此我们的建议对于诊断除 T1D 之外的各种其他自身免疫性疾病具有重要作用。公众健康相关性：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，在以后的生活中出现毁灭性并发症的风险很高。自身抗体是最强大的疾病生物标志物，我们的建议将开发改进的测量胰岛素自身抗体的程序，目前该程序的再现性较差。胰岛素是 1A 型糖尿病（尤其是幼儿）的主要分子靶点之一，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。