New Assay for MRBC-Specific Autoantibody Responses

MRBC 特异性自身抗体反应的新检测方法

• 批准号: 7315232
• 负责人: Catherine E Calkins
• 金额: $ 7.75万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-05 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7315232
• 关键词: Address; Age-Months; Animal Model; Antibodies; Antibody Affinity; Antigen Targeting; Antigens; Appearance; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Process; Autoimmune Responses; Autoimmune hemolytic anemia; Autoimmunity; B-Lymphocytes; Binding; Binding Sites; Biological Assay; Biotin; Cell Communication; Cells; Cellular Structures; Complex; Controlled Study; Coombs' Test; DNA Sequence; Detection; Development; Disease; Enzyme-Linked Immunosorbent Assay; Epitopes; Erythrocyte Membrane; Erythrocytes; Erythroid; Event; Failure; Flow Cytometry; Future; Genes; Genetic; Goals; Human; Immunoglobulin-Secreting Cells; Immunology; Inbred NZB Mice; Incidence; Indium; Individual; Integral Membrane Protein; Investigation; Lead; Mediating; Modeling; Mus; Pathologic; Pathology; Pattern; Penetrance; Peptides; Phage Display; Pilot Projects; Production; Random Peptide Libraries; Reaction; Regulation; Self Tolerance; Specificity; Staging; Study models; T-Lymphocyte; Technology; Testing; Therapeutic; Therapeutic Intervention; Work; assay development; base; enzyme linked immunospot assay; extracellular; middle age; mouse model; precursor cell; prevent; protein aminoacid sequence; research study; response; therapeutic target

DESCRIPTION (provided by applicant): The NZB mouse model of autoimmune hemolytic anemia was one of the first animal models of autoimmunity and has been important in contributing to our understanding of genetic control of anti-self responses and some of the cell interactions involved in these responses. The 100% incidence of disease in the NZB mice and their ability to control this response until middle age allows study of the model prior to, as well as after initiation of specific autoantibody production. Yet, the mechanisms of induction of the anti-erythrocyte autoantibodies in this mouse model are not known. Cellular studies of the controlling elements in this response have so far been limited by the use of an intact erythrocyte as the target antigen, for which assays of specific responses are complex and not very sensitive for low affinity antibodies. The use of target peptide MRBC epitopes could simplify and enhance the sensitivity of assays for this autoimmune response, making possible mechanistic studies of this autoantibody reaction that have not so far been approachable. A major portion of the pathogenic autoantibodies produced in these mice bind to erythrocyte band 3, however the actual target epitopes have not been defined, and band 3, as an integral membrane protein is difficult to obtain with structural integrity. Therefore, we propose a pilot study to focus on identifying the specific band 3 target epitopes of the NZB autoantibodies and then using these epitopes to develop assays that will be usable to detect individual autoantibody responses to each target epitope. Linear epitopes will be identified by testing selected peptide sequences deduced from DNA sequences of the extracellular loops of the erythrocyte band 3 for binding to a panel of pathogenic NZB monoclonal autoantibodies as well as to polyclonal autoantibodies extracted from autoimmune NZB mice. Target conformational epitopes of band 3 will be identified as mimotopes using a phage display approach for epitope discovery. ELISPOT assays for epitope-specific autoantibody secreting cells (AFC) will be developed using the selected target peptide epitopes as detection probes for secreted antibody. These assays will then be used to determine the importance of identified band 3 epitopes or mimotopes at each stage of NZB disease, focusing on the earliest stage identifiable to determine which epitope target(s) is/are involved in the initiation of this autoimmunity. This pilot study should make possible future investigations into the regulation of each epitope-specific autoantibody response in normal as well as pre- autoimmune and actively autoimmune NZB mice and into potential causes of the failure of these regulatory mechanisms in autoimmune NZB mice. The ultimate goal of these investigations into regulatory mechanisms that control the anti-MRBC response in NZB mice is to open up and test therapeutic opportunities for human autoimmune disease. NZB mice provide an animal model of human autoimmune disease in which anti-self erythrocyte (MRBC) antibodies are spontaneously produced and detectable in mid-life. The goal of the proposed work is to identify the MRBC peptide targets of the anti-MRBC autoantibodies made by these mice and to use these target peptides to develop assays for the autoreactive cells. Such assays will facilitate studies of the mechanisms of control versus induction of these autoantibody responses that will lead ultimately to therapeutic interventions in human autoimmune disease.

描述（由申请人提供）：自身免疫性溶血性贫血的NZB小鼠模型是自身免疫性的最早动物模型之一，对于我们对抗自身反应的遗传控制以及这些反应中涉及的一些细胞相互作用的理解非常重要。 NZB小鼠中疾病的100％发生率及其控制这种反应的能力，直到中年允许在模型之前以及在开始特定自身抗体产生之前研究该模型。然而，尚不清楚该小鼠模型中抗致电细胞自身抗体的诱导机制。到目前为止，该反应中控制元素的细胞研究受到使用完整的红细胞用作靶抗原的限制，对于特定反应的测定很复杂，对低亲和力抗体的敏感性不太敏感。靶肽MRBC表位的使用可以简化和增强对这种自身免疫反应的测定的敏感性，从而使对这种自身抗体反应的机械性研究可能无法接近。这些小鼠中产生的致病自身抗体的主要部分与红细胞带3结合，但是尚未定义实际的靶向缩略图，并且带3条带作为整体膜蛋白很难获得结构完整性。因此，我们提出了一项试点研究，专注于识别NZB自身抗体的特定频段3目标表位，然后使用这些表位来开发可用于检测每个目标表位的单个自身抗体响应的测定法。线性表位将通过测试从红细胞条带3的DNA序列推导的选定肽序列，以结合一组致病性NZB单克隆自身抗体的病原体NZB，从自身免除NZB小鼠中提取的多性自身抗体。带有噬菌体显示方法以发现表位的靶标构象表位将被鉴定为模仿。 ELISPOT针对表位特异性自身抗体分泌细胞（AFC）的ELISPOT分析将使用选定的靶肽表位作为分泌抗体的检测探针开发。然后，这些测定将用于确定NZB疾病的每个阶段鉴定的带3个表位或模仿的重要性，重点是确定最早的阶段，以确定哪些表位目标与这种自身免疫性有关。这项试点研究应使未来对正常和自身免疫前和自身免疫性NZB小鼠的调节的调节，并在自身免疫NZB小鼠中这些调节机制失败。这些调查对控制NZB小鼠抗MRBC反应的调节机制的最终目标是开放并测试人类自身免疫性疾病的治疗机会。 NZB小鼠提供了人类自身免疫性疾病的动物模型，其中抗自身的红细胞（MRBC）抗体是自发产生和可在中期生产的。拟议工作的目的是确定这些小鼠制造的抗MRBC自身抗体的MRBC肽靶标，并使用这些靶肽来开发自动反应性细胞的测定法。这些测定法将促进对控制机制的研究与这些自身抗体反应的诱导机制，这些反应最终会导致人类自身免疫性疾病的治疗干预措施。