DEVELOPMENT OF A SALIVARY ASSAY FOR MEASURING PERIODONTAL DISEASE ACTIVITY

项目总结/摘要 牙周病（PD）是全球人类最常见的慢性炎症性疾病，根据 疾病控制和预防中心（CDC）影响47%的30岁或以上的美国成年人， >60%的65岁以上患者。1鉴于PD的病程以疾病的不连续模式为特征， 活动和不活动显示组织破坏的加剧和缓解，2-4是对 牙周病的临床表现是什么？牙周病的临床表现是什么？ 表达活动性/进展性疾病的风险升高。不幸的是，目前的准则和分类 仅依靠临床和放射学测量来识别现有疾病。虽然这些记录 一个很好的反映了病人的过去的历史，5，6他们未能提供有关当前状态的信息， 这是因为活动性疾病包括 组织破坏性过程（生物表型）6，8，9，其先于并随后触发所产生的临床 体征和症状（临床表型）。已经鉴定了许多PD的唾液生物标志物7，10，11和12。 目前有多种商业测定可用于临床，但大多数缺乏对组织的特异性 破坏和/或不适合现场护理（POC）实时测试。基质金属蛋白酶-8是 在唾液中最广泛记录的、丰富的生物标志物中， 评估组织破坏的范围和程度。8，9，12-14作为一种酶，它存在于活性和潜在的 （非活性）形式，其相对表达决定了活性期间组织分解的程度。 因此，我们假设活性MMP-8与总MMP-8的比率（MMP-8活性/MMP-8总）将构成 比总MMP-8更有意义的疾病活动性生物学测量。然而，衡量疾病的标准 由于缺乏可商购的“活性”MMP-8的抗体，迄今为止活性还不可能。 建立在我们团队成功开发疾病活动特异性单克隆抗体（DASMAB）的基础上 针对MMP-8，我们在这里建议利用这些独特的资产来开发针对MMP-8的靶向分子测定， 量化疾病活动。在第一阶段的提案中，我们计划开发和验证一个优化的DASMAB- 基于ELISA的检测方法通过以下目的：（1）鉴定和选择最佳mAb对（捕获和检测） 用于每种MMP-8形式以产生ELISA试剂盒原型，和（2）疾病活性的分析和临床验证 ELISA原型。成功开发和验证首个基于DASMAB的疾病活性测定 将先于临床POC诊断产品，不仅为临床医生提供量化 实时评估疾病进展的程度和速度，还评估对治疗的反应并预测 未来事件最有可能的结果这些技术创新将使临床医生能够做出更多 有效的结果驱动的决策和个性化的预防策略，以改善病人的护理。

Project Summary / Abstract Periodontal disease (PD) is globally the most common chronic inflammatory disease in humans, which according to the Centers for Disease Control and Prevention (CDC) affect 47% of U.S. adults 30 years of age or older and >60% of those over 65 years.1 Given that the course of PD is marked by a discontinuous pattern of disease activity and inactivity showing exacerbation and remission of tissue destruction,2-4 a critical challenge for clinicians is not detection of clinical features of periodontal disease, but rather identification of patients who have an elevated risk for expressing active/progressing disease. Unfortunately, current guidelines and classifications rely exclusively on clinical and radiographic measurements to identify existing disease. While these records are a good reflection of a patient’s past history,5, 6 they fail to provide information on the current status of active disease or identify individuals and sites at risk for future disease.4, 5 This is because active disease involves tissue destructive processes (biologic phenotype)6, 8, 9 that precede and subsequently trigger the resulting clinical signs and symptoms (clinical phenotype). A host of salivary biomarkers for PD have been identified7, 10, 11 and multiple commercial assays are currently available for use in the clinic, but most lack specificity for tissue destruction and/or are not amenable to point-of-care (POC) real-time testing. Matrix metalloproteinase-8 is amongst the most widely documented, abundant biomarkers in saliva, and uniquely suited to quantitatively assess the extent and degree of tissue destruction.8, 9, 12-14 As an enzyme, it is present in both active and latent (inactive) forms, the relative expression of which dictate the extent of tissue breakdown during active periods of disease.8, 9 Hence, we hypothesize that the ratio of active and total MMP-8 (MMP-8Active/MMP-8Total) will constitute a more meaningful biological measure of disease activity than total MMP-8. However, measures of disease activity to date have not been possible due to the lack of commercially available antibodies for “active” MMP-8. Building off our team’s success in development of Disease Activity Specific Monoclonal Antibody (DASMAB) against MMP-8, we propose here to leverage these unique assets to develop a targeted molecular assay for quantifying disease activity. In this Phase I proposal, we plan to develop and validate an optimized DASMAB- based ELISA assay through the following aims: (1) identify and select the best mAb pairs (capture and detection) for each MMP-8 form to produce ELISA kit prototypes, and (2) analytical and clinical validation of disease activity of ELISA prototypes. Successful development and validation of the first DASMAB-based disease activity assay will precede a clinical POC diagnostic product that will offer clinicians with not only the means to quantify the extent and estimate the rate of disease progression in real-time, but also assess response to therapy and predict the most likely outcome of future events. These technological innovations will allow clinicians to make more effective outcome-driven decisions and personalize preventive strategies to improve patient care.