A kinetics approach to the characterization of an IgM specific for the glycolipid asialo-GM1
A kinetics approach to the characterization of an IgM specific for the glycolipid asialo-GM1
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DOI：
10.1016/s0022-1759(98)00012-x
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发表时间：
1998-03-01
影响因子：
2.2
通讯作者：
Altman, E
中科院分区：
文献类型：
--
作者：
Harrison, BA;MacKenzie, R;Altman, E
通过表面等离子体共振（SPR）监测抗体与脂质体中糖脂的相互作用，研究了膜锚定糖脂蛋白识别的独特特征。当检测与鞘糖脂asialo-GM1 （Gal β 1-3GalMAcl β 1-4Gal β 1-4Glc β 1-1- 1神经酰胺）结合时，鉴定出几个属于TgM和IgG类的阳性杂交瘤。初步筛选的酶免疫分析法和薄层色谱法（TLC）和免疫染色表明，只有IgM型对这种鞘糖脂具有特异性。其中一个IgMs H2G10被纯化，并使用SPR技术进一步表征，该技术涉及抗体结合到脂质体asialo-GM1。该方法得到了相互作用的动力学常数和亲和常数，并证实了H2G10对asialo-GM1I末端半乳糖的特异性。有趣的是，由于四糖浓度与H2G10解离速率常数呈反比关系，抑制抗体与asialo-GM1脂质体结合的asialo-GM1四糖减少了抗体结合的总量，但增加了抗原-抗体相互作用的亲和力。我们认为，这种效应是由于四糖选择性抑制低价结合，从而促进抗体-碳水化合物的高亲和力相互作用。观察到在高抗原抗体比下也观察到较慢的解离速率常数支持这一解释。这些结果强调了动力学数据可以在促进和抑制高亲和相互作用（如涉及蛋白质和碳水化合物的相互作用）方面提供的见解。(C) 1998 Elsevier Science B.V.版权所有
The unique features of protein recognition of membrane-anchored glycolipids were investigated by surface plasmon resonance (SPR) monitoring of antibody interactions with glycolipids contained in liposomes. Several positive hybridomas belonging to the TgM and IgG classes were identified when tested for binding to the glycosphingolipid asialo-GM1 (Gal beta 1-3GalMAcl beta 1-4Gal beta 1-4Glc beta 1-1-Ceramide). Preliminary screening by enzyme immunoassay and thin layer chromatography (TLC) followed by immunostaining indicated that only those of the IgM type showed specificity for this glycosphingolipid. One of the IgMs, H2G10, was purified and further characterized using a SPR technique that involved antibody binding to liposomal asialo-GM1. This method generated kinetic and affinity constants for the interaction and confirmed the specificity of H2G10 for the terminal galactose of asialo-GM1I. Interestingly, inhibition of antibody binding to asialo-GM1 liposomes by the asialo-GM1 tetrasaccharide reduced the total amount of bound antibody but increased the affinity of the antigen-antibody interaction due to an inverse relationship between tetrasaccharide concentration and the H2G10 dissociation rate constant. We believe that this effect is due to the selective inhibition of lower valency binding by the tetrasaccharide which, in turn, promotes higher avidity antibody-carbohydrate interactions. The observation that slower dissociation rate constants were also observed at high antigen to antibody ratios supports this interpretation. These results highlight the insight that kinetic data can provide in efforts to promote and inhibit high avidity interactions such as those involving proteins and carbohydrates. (C) 1998 Elsevier Science B.V. Air rights reserved.