Oppliger I, Vaananen H K, Munoz P A, Hsu H H, Morris D C, Anderson H C
Bone. 1986;7(5):373-8. doi: 10.1016/8756-3282(86)90258-9.
Alkaline phosphatase (ALPase), concentrated in the membranes of matrix vesicles, is believed to play a role in initial calcification. To further purify, characterize, and identify this enzyme in tissue, a monoclonal antibody was developed against the ALPase of isolated fetal calf matrix vesicles. Splenic lymphocytes derived from mice immunized with Sepharose 6B-purified fetal calf matrix vesicle ALPase were fused with mouse plasmacytoma cells (line X63-Ag-8.653) using standard hybridoma technology. Hyperimmune sera and hybridoma culture supernatants were screened for the presence of specific antibody using a newly developed double-immunosorbent assay in which putative antibody is added to microtiter plate wells precoated with affinity-purified rabbit antimouse immunoglobulin. After incubation and washing, partially purified fetal calf matrix vesicle ALPase is added to each well. The enzyme adheres only to wells that contain specific anti-ALPase antibody. These wells are identified by adding the enzyme substrate p-nitrophenyl phosphate and reading the wells in a plate-reading spectrophotometer at 405 nm. A hybridoma-producing specific antibody was subsequently cloned and grown as ascities-producing tumors in pristane-primed mice. Ouchterlony analysis indicated that the cell line secretes an immunoglobulin of IgG1 class. This antibody reacts specifically with ALPase derived from calf matrix vesicles and cross-reacts with ALPase of bovine kidney, liver, and placental origin and human bone but does not cross-react with bovine intestinal ALPase or ALPase derived from matrix vesicles isolated from rachitic rat growth plate cartilage.
碱性磷酸酶（ALPase）集中于基质小泡的膜中，被认为在初始钙化过程中发挥作用。为了在组织中进一步纯化、表征和鉴定这种酶，制备了一种针对分离的胎牛基质小泡ALPase的单克隆抗体。使用标准杂交瘤技术，将用琼脂糖6B纯化的胎牛基质小泡ALPase免疫的小鼠脾脏淋巴细胞与小鼠浆细胞瘤细胞（X63-Ag-8.653系）融合。使用新开发的双免疫吸附测定法筛选超免疫血清和杂交瘤培养上清液中是否存在特异性抗体，该测定法是将假定的抗体加入预先用亲和纯化的兔抗小鼠免疫球蛋白包被的微量滴定板孔中。孵育和洗涤后，将部分纯化的胎牛基质小泡ALPase加入每个孔中。该酶仅粘附于含有特异性抗ALPase抗体的孔中。通过加入酶底物对硝基苯磷酸酯并在405nm处用酶标仪读取孔来鉴定这些孔。随后克隆产生特异性抗体的杂交瘤，并在预先用 pristane 处理的小鼠中作为产生腹水的肿瘤生长。双向免疫扩散分析表明，该细胞系分泌IgG1类免疫球蛋白。这种抗体与源自小牛基质小泡的ALPase特异性反应，并与牛肾、肝和胎盘来源以及人骨的ALPase交叉反应，但不与牛肠ALPase或源自佝偻病大鼠生长板软骨分离的基质小泡的ALPase交叉反应。
