基于NAPPA技术的梅毒预后监测血清标志物筛选及应用评估

The efficacy evaluation for syphilis treatment is very difficult to conduct in clinical practice so far, and still no solid cure criteria for syphilis prognosis surveillance. Our preliminary experiment results indicated that different Treponema pallidum (Tp) antigens and corresponding antibodies presented time-related feature in the course of syphilis treatment and these variations are closely associated with the treatment efficacy. Hypothesis that the changes of all 1039 Treponema pallidum proteins and their antibodies can be monitored in the serum before and after treatment, it is expected to find novel target for syphilis prognosis surveillance. Aiming to validate the hypothesis, we will conduct following works: 1) Conduct Tp cDNA library preparation and construct Nucleic Acid Programmable Protein Aarray (NAPPA）chip antibody detection technique, 2) By utilizing the NAPPA chip to analysis antibody expression profiles from different syphilis patients and to obtain the information on antibodies and the corresponding antigens association with the treatment efficacy,3) By applying immunological methods to verity the antibodies associated with prognosis monitoring and to further screen the antibody serum markers,4) By applying immunological methods to verity the antigens related to prognosis monitoring and further screen the antigen serum markers,5）By employing decision tree algorithm to identify the optimal combination of serum markers for prognosis monitoring, 6) to establish Suspension Array Technology for monitoring syphilis prognosis and to conduct the assessment on its clinical application. The present study breaks the conventional idea that syphilis-specific antibodies can not be used for monitoring treatment efficacy. It would be a breakthrough to provide solution to this long-plagued problems in clinical practice and syphilis prognosis surveillance.

至今梅毒的疗效评价十分困难，缺乏临床治愈判断标准。我们发现：血清中梅毒螺旋体（Tp）抗原及其抗体在治疗过程中呈时相性变化，且部分指标与疗效相关。假设能监测血清中全部1039个Tp蛋白质及其抗体在治疗前后的变化，将有望找到预后监测新靶点。本项目拟开展：1）建立Tp cDNA文库，构建NAPPA芯片Tp抗体检测技术；2）采用NAPPA芯片技术研究不同梅毒患者血清抗体表达谱，初筛获得与疗效相关抗体，以及对应的靶抗原信息；3）免疫学方法验证与预后监测相关的抗体，进一步筛选血清抗体标志物；4）免疫学方法验证与预后监测相关的抗原，进一步筛选血清抗原标志物；5）采用决策树分析法建立最佳的预后监测血清标志物的组合模型；6）构建梅毒预后监测的液态芯片技术检测预后检测指标，并开展临床应用评估。本研究打破传统“梅毒特异性抗体不能用于疗效监测”的观念，筛选获得梅毒预后监测血清标志物，解决长期困扰临床的盲区与难点。

近20 多年来，梅毒一直是我国乃至世界范围的重要社会公共健康问题，其发病数逐年上升，位居性传播性疾病的首位。尽管青霉素应用于治疗和改善各类型梅毒已经超过半个世纪，但是至今无论采用哪种推荐的治疗方案都有可能导致治疗失败，评价其疗效十分困难，缺乏有效的判断标准。精确诊断是科学处理的前提，寻找在最适时间内能对患者作出准确疗效判断的生物学标志物，是梅毒治疗中亟待解决的问题。近年来，人们聚焦于寻找覆盖全病程的高免疫原性蛋白质，并以其为靶抗原成功构建了诸多高灵敏性的诊断试剂盒，但不能用于疗效监测。我们采用蛋白组学技术分析梅毒治愈和梅毒非治愈患者的Tp抗原表达差异谱，共获得24个蛋白，采用聚类分析进一步筛选梅毒治愈组的差异蛋白，发现R9UUE6、R9UVV0、R9USL8、A0A386TQZ6、R9UY59、R9URW7和R9UV19共 7个蛋白在治愈组的表达明显低于非治愈组。采用临床样本对所筛选的差异蛋白进行验证，梅毒治愈组的R9UUE6、R9USL8、A0A386TQZ6、R9UY59和R9UV19 的OD值明显低于梅毒非治愈组，两组R9UVV0和R9URW7的OD值没有差异。构建的R9UUE6、R9USL8、A0A386TQZ6、R9UY59和R9UV19多靶液态芯片检测技术用于差异蛋白的检测，并对构建的液态芯片检测技术进行临床应用研究，多靶液态芯片检测技术诊断梅毒血清固定治愈的灵敏度为81.33%，特异性为78.00%。为梅毒治愈提供新的指标。但其检测的灵敏度和特异性还有待进一步提高。