Arklie J, Trowsdale J, Bodmer W F
Tissue Antigens. 1981 Mar;17(3):303-12. doi: 10.1111/j.1399-0039.1981.tb00705.x.
A monoclonal antibody (AAP1) to human intestinal alkaline phosphatase (ALP) was produced by immunizing a mouse with D98/AH-2 (HeLa) cells, which produce the enzyme ectopically. The antibody, which did not inhibit enzyme activity using p-nitrophenyl phosphate as the substrate, was of the IgG2A class and did not show complement-dependent cytotoxicity. In trace binding assays AAP1 bound only to cells that expressed an intestinal-like form of human ALP, including some human intraspecific (D98/AG-2 x human lymphocyte or fibroblast) hybrids. Immunoprecipitation of immune complexes from cell-free extracts of D98/AG-2 cells, using protein A containing S. aureus and AAP1 antibody, resulted in precipitation of all the ALP activity. The precipitated material had a subunit molecular weight of 80,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. In non-denaturing conditions, AAP1 antibody prevented the migration of ALP activity into the gel when cell-free extracts were made from human adult or fetal intestine, or D98/AH-2 cells. Similarly, AAP1 could be used to precipitate ALP activity from these extracts but not from extracts of human liver, kidney or placenta.
用异位产生该酶的D98/AH-2（海拉）细胞免疫小鼠，制备了一种针对人肠碱性磷酸酶（ALP）的单克隆抗体（AAP1）。该抗体以对硝基苯磷酸酯作为底物时不抑制酶活性，属于IgG2A类，且不显示补体依赖性细胞毒性。在微量结合试验中，AAP1仅与表达人ALP肠样形式的细胞结合，包括一些人种内（D98/AG-2×人淋巴细胞或成纤维细胞）杂种细胞。使用含金黄色葡萄球菌的蛋白A和AAP1抗体，对D98/AG-2细胞的无细胞提取物中的免疫复合物进行免疫沉淀，结果沉淀出了所有的ALP活性。经十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳估计，沉淀物质的亚基分子量为80,000道尔顿。在非变性条件下，当从成人或胎儿小肠或D98/AH-2细胞制备无细胞提取物时，AAP1抗体可阻止ALP活性迁移到凝胶中。同样，AAP1可用于从这些提取物中沉淀ALP活性，但不能从人肝脏、肾脏或胎盘的提取物中沉淀。
