Haavik S, Codington J F, Davison P F
Department of Pharmacognosy, Institute of Pharmacy, Oslo, Norway.
Glycobiology. 1992 Jun;2(3):217-24. doi: 10.1093/glycob/2.3.217.
The preparation of greater than 30 different hybridomas, all secreting IgM class antibodies against epiglycanin, a glycoprotein at the surface of the mouse mammary carcinoma cell line TA3-Ha, is described. The specificities of 10 of the antibodies, with affinity constants in the range of 10(8)-10(10) l/mol were compared in an enzyme competitive binding assay. The affinity of epiglycanin was strongly reduced for all antibodies tested by incubation with periodate (10 mM, 4 degrees C) and was reduced for most of the antibodies by endo-alpha-N-acetyl- D-galactosaminidase. This suggested that carbohydrate, and specifically the Gal beta (1----3)GalNAc disaccharide, formed an integral part of the epitopes of most of the antibodies. The isolated disaccharide, however, exhibited 250,000 times less inhibitory activity in the competitive binding assay than epiglycanin. The binding capacity of epiglycanin was also reduced by incubation with trypsin or pronase, suggesting a high molecular weight dependency for binding. Incubation with sialidase increased its affinity for the antibodies. The binding of the antibodies to epiglycanin was strongly inhibited by peanut agglutinin, and to a lesser extent by lectins from Triticum vulgaris, Ricinus communis, Pisum sativum and Phaseolus vulgaris. None of the antibodies bound to any of eight different gangliosides immobilized on HPTLC plates. Mono- (Fab) and divalent [F(ab')2] fragments of the antibodies possessed very low affinity for epiglycanin. The results demonstrated that the specificities of the antibodies are related, but distinguishable, and they suggest that this epiglycanin-IgM model may be useful for studies on the general principles of the interaction between IgM antibodies and mucin-type glycoproteins.
本文描述了30多种不同杂交瘤的制备方法，这些杂交瘤均分泌针对小鼠乳腺癌细胞系TA3-Ha表面糖蛋白表位糖蛋白的IgM类抗体。在酶竞争性结合试验中比较了其中10种抗体的特异性，其亲和常数在10(8)-10(10) l/mol范围内。通过与高碘酸盐（10 mM，4℃）孵育，所有测试抗体对表位糖蛋白的亲和力均显著降低，并且大多数抗体经内切α-N-乙酰-D-半乳糖胺酶处理后亲和力也降低。这表明碳水化合物，特别是Galβ(1----3)GalNAc二糖，构成了大多数抗体表位的一个组成部分。然而，在竞争性结合试验中，分离出的二糖的抑制活性比表位糖蛋白低250,000倍。用胰蛋白酶或链霉蛋白酶孵育也会降低表位糖蛋白的结合能力，这表明结合具有高分子量依赖性。用唾液酸酶孵育可增加其对抗体的亲和力。抗体与表位糖蛋白的结合受到花生凝集素的强烈抑制，而受到来自普通小麦、蓖麻、豌豆和菜豆的凝集素的抑制作用较小。没有一种抗体与固定在高效薄层层析板上的八种不同神经节苷脂中的任何一种结合。抗体的单（Fab）和二价[F(ab')2]片段对表位糖蛋白的亲和力非常低。结果表明这些抗体的特异性相关但可区分，并且表明这种表位糖蛋白-IgM模型可能有助于研究IgM抗体与粘蛋白型糖蛋白相互作用的一般原理。
