Klaamas K, Kodar K, Kurtenkov O
National Institute for Health Development, Tallinn, Estonia.
Neoplasma. 2008;55(2):143-50.
All human immunoglobulins are glycosylated. The changes in IgG glycosylation are associated with autoimmune disorders and pregnancy. Little is known about IgG glycosylation in patients with cancer. A lectin enzyme-linked immunosorbent assay (LELISA) based method was developed for measuring the Concanavalin A - positive IgG in the serum. Its rationale is as follows: PtA was used as a capture agent for binding IgG via the Fc fragment. Then IgG and the ConA-positive glycans on the IgG were detected using an anti-human IgG-F(ab)2 alkaline phosphatase conjugate or biotinylated ConA, respectively. The index ConA binding/total IgG was calculated. Serum samples from patients with gastric carcinoma (n=53) and healthy blood transfusion donors (n=24) were analysed. The protein A-agarose and ConA-sepharose affinity chromatography was applied to the purification of IgG, ConA-positive IgG, and Fab fragments. The LELISA, SDS-PAGE and Western blot methods were used to analyse the purified IgG and Fab fragments. A significantly higher ConA binding to IgG was found in patients with cancer compared to that of blood donors (ConA index = 1.07+/-0.08 (95% CI) and 0.81+/-0.08, respectively; P=0.0002). In donors, a significant correlation between the level of IgG bound to PtA and the ConA binding (r=0.85; p<0.001) was observed. Patients with gastric cancer showed a less pronounced, though significant correlation (r=0.33; P=0.02). Only the Fd fragment of the Fabs derived from both total serum IgG and ConA-positive fraction of IgG contained the ConA-positive glycans. The comparison of the purified IgG and Fab fragments derived from healthy blood donors and patient with gastric cancer showed no difference in either SDS-PAGE, immunoblotting or LELISA pattern. The LELISA is simple, reproducible and suitable for the evaluation of IgG glycosylation changes. The level of ConA positive serum IgG was found to be increased in patients with cancer. No convincing evidence of the presence of asymmetrically glycosylated F(ab)2 fragments was found. A trend towards a better survival of patients with a lower level of the ConA-positive IgG was observed suggesting a possible blocking effect of the latter on tumor immunity.
所有人类免疫球蛋白都进行了糖基化修饰。IgG糖基化的变化与自身免疫性疾病及妊娠相关。关于癌症患者的IgG糖基化情况，人们了解甚少。开发了一种基于凝集素酶联免疫吸附测定（LELISA）的方法来检测血清中刀豆球蛋白A阳性IgG。其原理如下：使用蛋白A作为捕获剂，通过Fc片段结合IgG。然后分别使用抗人IgG-F(ab)2碱性磷酸酶结合物或生物素化刀豆球蛋白A检测IgG及其上的刀豆球蛋白A阳性聚糖。计算刀豆球蛋白A结合/总IgG指数。分析了胃癌患者（n = 53）和健康输血者（n = 24）的血清样本。采用蛋白A-琼脂糖和刀豆球蛋白A-琼脂糖亲和色谱法纯化IgG、刀豆球蛋白A阳性IgG和Fab片段。使用LELISA、SDS-PAGE和Western印迹法分析纯化后的IgG和Fab片段。与输血者相比，癌症患者中刀豆球蛋白A与IgG的结合显著更高（刀豆球蛋白A指数分别为1.07±0.08（95%置信区间）和0.81±0.08；P = 0.0002）。在输血者中，观察到与蛋白A结合的IgG水平与刀豆球蛋白A结合之间存在显著相关性（r = 0.85；p < 0.001）。胃癌患者也显示出相关性，尽管不太明显（r = 0.33；P = 0.02）。来自总血清IgG和IgG的刀豆球蛋白A阳性部分的Fabs仅Fd片段含有刀豆球蛋白A阳性聚糖。对来自健康输血者和胃癌患者的纯化IgG和Fab片段进行比较，在SDS-PAGE、免疫印迹或LELISA模式上均未发现差异。LELISA方法简单、可重复，适用于评估IgG糖基化变化。发现癌症患者血清中刀豆球蛋白A阳性IgG水平升高。未发现存在不对称糖基化F(ab)2片段的令人信服的证据。观察到刀豆球蛋白A阳性IgG水平较低的患者有更好生存的趋势，提示后者可能对肿瘤免疫有阻断作用。
