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

在本研究中，从健康志愿者的混合血清中高度纯化了免疫球蛋白G。然后，通过酶促低聚糖改造，从人的免疫球蛋白中制备无半乳糖的免疫球蛋白。对完整的免疫球蛋白和低聚糖修饰的免疫球蛋白的蛋白质部分和寡糖结构的分析表明，低聚糖修饰完全去除了免疫球蛋白中的半乳糖残基，而不破坏蛋白部分。利用这种无半乳糖的免疫球蛋白，设计了检测类风湿因子的新的血清检测体系如下。将不含半乳糖的免疫球蛋白固定在96孔板上，加入血清样品。如果血清样本中存在类风湿因子，它就会与平板上的无半乳糖抗体结合。用HRP标记凝集素检测与无半乳糖抗体结合的类风湿因子的半乳糖残留量。考察了凝集素、去污剂、封闭剂的种类和浓度、血清样品的分析量、反应温度、反应时间等因素对固定化无半乳糖免疫球蛋白的条件。最后，建立了检测类风湿关节炎患者血清中类风湿因子寡糖的最佳检测条件。通过对健康人、类风湿性关节炎、风湿性疾病和其他疾病患者的血清标本进行分析，结果表明，在类风湿关节炎患者(包括血清阴性患者和早期患者)的特异性检测和了解疾病活动性方面，这种新的血清检测方法优于传统的血清试验(RA试验和RAHA试验)。因此，这一新的血清检测方法对该病的准确诊断和治疗是非常有用的。

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.