Mårtensson S, Due C, Påhlsson P, Nilsson B, Eriksson H, Zopf D, Olsson L, Lundblad A
Department of Clinical Chemistry, University Hospital, Lund, Sweden.
Cancer Res. 1988 Apr 15;48(8):2125-31.
A monoclonal antibody, 43-9F, specifically recognizes a tumor-associated antigen expressed both on surface membrane glycoproteins and on secreted soluble mucins of human squamous lung carcinoma (SLC) cells, and the corresponding antigen can be detected as a circulating tumor marker in plasma of SLC patients. Thin-layer chromatography immunostaining of neutral glycolipids extracted from SLC cells reveals a 43-9F-reactive glycolipid whose carbohydrate structure, as determined by fast atom bombardment-mass spectrometry, is identical with that of an Lea-active pentaglycosylceramide described previously: Gal beta 1-3[Fuc alpha 1-4]-GlcNAc beta 1-3Gal beta 1-4Glc-Cer. However, the Lea-active oligosaccharide hapten, lacto-N-fucopentaose II, with the same carbohydrate structure, fails to inhibit binding of 43-9F, and a well-characterized anti-Lea monoclonal antibody blocks only 40% of 43-9F binding sites on SLC cells, suggesting that the major epitope recognized by 43-9F is more complex than the Lea epitope. To search for a higher affinity 43-9F epitope among more complex oligosaccharides, a mixture of tritiated neutral oligosaccharide alditols from pooled human milk was passed through a 43-9F affinity column. A major retarded oligosaccharide was purified by high-performance liquid chromatography and shown by fast atom bombardment-mass spectrometry to have the following structure: Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4[Fuc alpha 1-3] GlcNAc beta 1-3Gal beta 1-4Glc. Oligosaccharides containing this sugar sequence are at least 100-fold more active than lacto-N-fucopentaose II as competitive inhibitors of 43-9F. Thus, antibody 43-9F binds to the above difucosyl Lea-X determinant with high affinity and weakly cross-reacts with the Lea antigen under some conditions such as occurs in thin-layer chromatography and enzyme-linked immunosorbent assay where multiple weak interactions of the decavalent IgM antibody may occur.
一种单克隆抗体43 - 9F能特异性识别一种肿瘤相关抗原，该抗原在人肺鳞状细胞癌（SLC）细胞的表面膜糖蛋白和分泌的可溶性粘蛋白上均有表达，并且相应抗原可在SLC患者血浆中作为循环肿瘤标志物被检测到。对从SLC细胞中提取的中性糖脂进行薄层色谱免疫染色，发现一种与43 - 9F反应的糖脂，通过快原子轰击质谱法测定其碳水化合物结构，与先前描述的Lea活性五糖基神经酰胺相同：Galβ1 - 3[Fucα1 - 4] - GlcNAcβ1 - 3Galβ1 - 4Glc - Cer。然而，具有相同碳水化合物结构的Lea活性寡糖半抗原乳糖 - N - 岩藻五糖II不能抑制43 - 9F的结合，且一种特征明确的抗Lea单克隆抗体仅能阻断SLC细胞上43 - 9F结合位点的40%，这表明43 - 9F识别的主要表位比Lea表位更复杂。为了在更复杂的寡糖中寻找与43 - 9F具有更高亲和力的表位，将来自混合人乳的氚标记中性寡糖醇混合物通过43 - 9F亲和柱。一种主要的滞留寡糖通过高效液相色谱法纯化，并通过快原子轰击质谱法显示具有以下结构：Galβ1 - 3[Fucα1 - 4]GlcNAcβ1 - 3Galβ1 - 4[Fucα1 - 3]GlcNAcβ1 - 3Galβ1 - 4Glc。含有该糖序列的寡糖作为43 - 9F的竞争性抑制剂，其活性比乳糖 - N - 岩藻五糖II至少高100倍。因此，抗体43 - 9F以高亲和力结合上述二岩藻基Lea - X决定簇，并且在某些条件下，如在薄层色谱和酶联免疫吸附测定中可能发生十价IgM抗体的多种弱相互作用时，与Lea抗原发生弱交叉反应。
