U UREALYTICUM ANTIGEN

描述(改编自申请者摘要)：解脲支原体是一种 常见的泌尿生殖道共生现象，然而，它是导致 早产儿绒毛膜羊膜感染与发病率和死亡率。 对人类免疫反应中重要的抗原知之甚少。 有14个已知的血清型，可分为两个生物型。 据推测，只有特定的血清型或单一的 生物型可能会导致侵袭性疾病，而缺乏血清型或 生物型特异性抗体可能是一个重要的危险因素。现在 资助金申请是在#年提交的资助金首次竞争性续期 对RFA 88-AI-12的响应，其目标是开发单克隆化 用于表位映射的抗体(单抗)以识别血清型或 血清群特异性抗原。调查员和同事之前 在解脲支原体上发现了一个抗原复合体，命名为MB，这表明 血清型和/或血清组的特异性是在表位而不是 全抗原水平。在这一授权期内，他们展示了 在所有14个血清型上存在MB；该抗原包含这两个血清型 和交叉反应表位；MB不仅在体外产生，而且 在体内；MB是解脲支原体最主要的识别抗原之一 人类感染；抗MB单抗可防止发生 疾病。通过抗体反应肽扫描(PepScan)，他们已经显示 MB血清型3和血清型的主要免疫反应部位 特异性映射到羧基区域。使用PepScan分析，他们拥有 建立了一种合成肽酶免疫分析(EIA)，将允许 确定抗体对最具抗原性表位的反应性(S) 在解脲支原体感染患者的血清中。重要的是，他们有 证实了羧基结构域的独特结构允许 导致MB抗原大小变化的突变。他们已经展示了 羧基末端由串联的6个氨基酸重复组成 在不同的临床分离株中发生，最少7个拷贝，最多 是42份。调查人员表示，所有迹象表明，这是 变异可能是疾病产生的关键决定因素。他们 已经开发出了聚合酶链式反应引物，可以检测出大小的变异 已经开发出可靠的聚合酶链式反应技术作为检测的培养物 患者标本中解脲支原体的感染。本补助金申请的具体目的 将：(1)从剩余的13个UU血清型中克隆MB并对其进行测序 在本赠款期间建立的方法；(2)开发聚合酶链式反应方法 检测体内的血清型和MB大小变异；以及(3)通过PepScan建立 其余13个毒株的血清型和生物型特异性表位 并使用基于合成肽的EIA来确定血清变量， 生物群和总MB抗体(包括同种和亚类)反应 在有记录的侵袭性解脲支原体感染的患者中。抗体图谱将会 与患者中存在的血清型(S)和大小变异(S)进行比较 用聚合酶链式反应确定的位点。这些研究将建立、表征和 验证调查人员认为应该被证明有用的试剂 不同血清型和生物型在肉鸡生产中的作用 侵袭性解脲支原体疾病及血清特异性抗体在解脲支原体感染中的作用 保护。

DESCRIPTION (Adapted from applicant's abstract): U. urealyticum is a common commensal of the urogenital tract, yet, it is an important cause of chorioamnion infection and morbidity and mortality in premature infants. Antigens important in the human immune response are poorly understood. There are 14 recognized serovars which can be divided into two biotypes. It has been postulated that only certain serovars or members of a single biotype may be able to cause invasive disease and that lack of serovar or biotype specific antibody may be an important risk factor. The present grant application is the first competitive renewal of a grant submitted in response to RFA 88-AI-12 the goal of which was to develop monoclonal antibodies (mAbs) to be used in epitope mapping to identify serovar or serogroup specific antigens. The investigator and colleagues previously identified an antigen complex on Uu, designated MB, which suggested that serovar- and/or serogroup specificity is at the epitope rather than the whole antigen level. During this grant period, they have shown the existence of MB on all 14 serovars; that this antigen contains both serovar and cross-reactive epitopes; that MB is produced not only in vitro but also in vivo; MB is one of the most predominant antigens recognized during Uu infection of humans; and mAbs to MB can protect against development of disease. By antibody-reactive peptide scanning (Pepscan), they have shown that the major immunoreactive site of MB serovar 3 as well as serovar specificity maps to the carboxy region. Using Pepscan analysis they have established a synthetic peptide enzyme immunoassay (EIA) which will allow them to determine the antibody reactivity to the most antigenic epitope(s) in sera of ureaplasma infected patients. Importantly, they have established that the unique structure of the carboxy domain allows mutations that result in size variation of the MB antigen. They have shown that the carboxy terminus is composed of concatemeric 6 amino acid repeats occurring in different clinical isolates in as few as 7 copies and as many as 42 copies. The investigator states that all indications are that this variation may be a critical determinant in production of disease. They have developed PCR primers which allow detection of size variants plus they have developed PCR techniques which are reliable as culture for detection of Uu in patient specimens. Specific aims of the present grant application are to: (1) clone and sequence MB from the remaining 13 Uu serovars using methods established in the present grant period; (2) develop PCR methods to detect serovars and MB size variants in vivo; and (3) establish by Pepscan the serovar and biotype specific epitopes of each of the remaining 13 serovars; and use a synthetic peptide-based EIA to determine the serovar, biogroup, and total MB antibody (including isotype and subclass) response in patients with documented invasive Uu infection. Antibody profiles will be compared to the serovar(s) and size variant(s) present in the affected site as determined by PCR. These studies will establish, characterize, and validate reagents which the investigators feel should prove useful in elucidating the role of different serovars and biotypes in production of invasive Uu disease and the role of serovar specific antibody in protection.