Karlsson G, Månsson J E, Wikstrand C, Bigner D, Svennerholm L
Department of Psychiatry and Neurochemistry, Gothenburg University, St. Jörgen Hospital, Hisings Backa, Sweden.
Biochim Biophys Acta. 1990 Apr 17;1043(3):267-72. doi: 10.1016/0005-2760(90)90026-t.
Several derivatives of ganglioside GM2 were synthesized for mapping of the binding epitope of a monoclonal antibody raised against this ganglioside. The GM2 ganglioside was modified in both the hydrophobic and the hydrophobilic part of the molecule. The synthesized derivatives were characterized with fast atom bombardment mass spectrometry (FAB-MS). Affinity of the monoclonal antibody for the GM2 derivatives was determined by enzyme-linked immunosorbent assay (ELISA) on microtitre plates or by TLC immunostaining. Modifying the GM2 sialic acid by deacetylation or blocking of the carboxyl moiety abolished the binding to the monoclonal antibody while the cleaving of the glycol group on the sialic acid tail led to a 70% reduced binding affinity. Removal of the fatty acid (lyso-GM2) eliminated the binding to the antibody. GM2 derivatives with fatty acid moieties of 8 carbon atoms or less showed almost no reactivity. GM2 with saturated fatty acids 16:0, 18:0 and 20:0 had binding affinity similar to natural GM2, while the 24:0 fatty acid had only half the binding affinity. The results demonstrate the importance of ganglioside fatty acid composition with regard to ligand binding between the monoclonal antibody and its specific ganglioside antigen. Thus, caution must be shown in the application of immunaffinity methods with monoclonal antibodies for the quantitative determination of glycosphingolipids from different tissues.
合成了神经节苷脂GM2的几种衍生物，用于绘制针对该神经节苷脂产生的单克隆抗体的结合表位图谱。GM2神经节苷脂在分子的疏水和亲水部分均进行了修饰。合成的衍生物通过快原子轰击质谱法（FAB-MS）进行表征。单克隆抗体对GM2衍生物的亲和力通过微量滴定板上的酶联免疫吸附测定（ELISA）或薄层层析免疫染色来确定。通过去乙酰化或封闭羧基部分修饰GM2唾液酸会消除与单克隆抗体的结合，而切割唾液酸尾部的糖基会导致结合亲和力降低70%。去除脂肪酸（溶血GM2）会消除与抗体的结合。具有8个或更少碳原子脂肪酸部分的GM2衍生物几乎没有反应性。具有饱和脂肪酸16:0、18:0和20:0的GM2具有与天然GM2相似的结合亲和力，而24:0脂肪酸的结合亲和力只有一半。结果证明了神经节苷脂脂肪酸组成对于单克隆抗体与其特异性神经节苷脂抗原之间配体结合的重要性。因此，在应用单克隆抗体免疫亲和方法定量测定不同组织中的糖鞘脂时必须谨慎。
