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Development of methods for specific detection of oligosaccharide chains of rheumatoid factor in sera from patients with early-phase rheumatoid arthritis.

开发早期类风湿关节炎患者血清中类风湿因子寡糖链的特异性检测方法。

基本信息

批准号：
07557226
负责人：
MIZUOCHI Tsuguo
金额：
$ 3.01万
依托单位：
Tokai University, School of Engineering
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

In the present study, IgG was highly purified from pooled sera of healthy volunteers. Galactose-free IgG was then prepared from the human IgG by enzymatic oligosaccharide-remodelling. Analyzes of the protein moiety and oligosaccharide structures of both intact IgG and the oligosaccharide-remodelled IgG indicated that the oligosaccharide-remodelling completely removed galactose residues of IgG without damaging the protein moiety. Using this galactose-free IgG,novel serum assay system for detection of rheumatoid factor was designed as follows. The galactose-free IgG was immobilized on a 96-well microtiter plate, to which serum sample was added. If rheumatoid factor exists in the serum sample, it binds to galactose-free IgG on the plate. Them galactose residues of rheumatoid factors bound to the galactose-free IgG were detected with HRP conjugated lectin. Conditions for immobilizing the galactose-free IgG on the plate, a kind and concentration of the lectin, detergent, and blocking reagent, amount of serum sample analyzed, reaction temperature, and reaction time were investigated in detail. Finally, an optimal assay condition was established for specific detection of oligosaccharides of rheumatoid factor in sera from patients with rheumatoid arthritis. When serum samples from healthy individuals, patients with rheumatoid arthritis, rheumatic diseases, and the other diseases were analyzed using this novel serum test, it was indicated that this novel serum test is superior to conventional serum tests (RA test and RAHA test) for detection of rheumatoid factor in terms of specific detection of patients with rheumatoid arthritis including seronegative patients and early-phase patients and understanding of the disease activity in patients. Therefore, this novel serum test could be very useful for accurate diagnosis and treatment of this disease.

在本研究中，IgG从健康志愿者的汇集血清中高度纯化。通过酶促低聚糖重塑法制备无半乳糖IgG。对完整IgG和低聚糖重塑IgG的蛋白质片段和低聚糖结构的分析表明，低聚糖重塑完全去除了IgG的半乳糖残基，而不破坏蛋白质片段。利用该无半乳糖IgG，设计了一种检测类风湿因子的新型血清检测系统。将无半乳糖IgG固定在96孔微滴板上，加入血清样品。如果血清样本中存在类风湿因子，它会与平板上无半乳糖的IgG结合。用HRP偶联凝集素检测与无半乳糖IgG结合的类风湿因子的半乳糖残基。考察了无半乳糖IgG在平板上固定的条件、凝集素、洗涤剂、阻断剂的种类和浓度、血清样品的分析量、反应温度和反应时间。最后，建立了类风湿关节炎患者血清中类风湿因子低聚糖特异性检测的最佳检测条件。对健康人、类风湿关节炎患者、风湿性疾病患者及其他疾病患者的血清样本进行分析，结果表明，在对类风湿关节炎患者（包括血清阴性患者和早期患者）的特异性检测和对患者疾病活动度的了解方面，该新型血清检测方法优于常规血清检测方法（RA试验和RAHA试验）。因此，这种新的血清检测方法对该病的准确诊断和治疗具有重要意义。