Durbin H, Tucker D F, Milligan E M, Bobrow L G, Warne P H, Pookim Y L, Bodmer W F
Imperial Cancer Research Fund, London, UK.
Int J Cancer Suppl. 1988;2:50-8. doi: 10.1002/ijc.2910410714.
We describe the production and preliminary characterisation of a set of monoclonal antibodies (MAbs) raised against placental alkaline phosphatase (PLAP). Different forms of antigen presentation, PLAP or PLAP-like expressing whole cells, placental membranes or purified PLAP were used to immunise BALB/c mice. Initial screening was carried out against the immunising material by ELISA, against fresh frozen placental sections by immunostaining and against purified PLAP using an enzyme capture assay. The most successful fusions were those following whole cell immunisation, producing 27 antibodies that all reacted with both the placental and testicular form of enzyme. These all showed a broadly similar pattern of reactivity when tested against a range of human malignant cell lines. Further characterisation identified one antibody, 8B6, as strongly reactive with formalin-fixed paraffin-embedded placental sections. This antibody also performed well when tested against a range of normal and malignant routinely fixed tissue sections. Of 14 antibodies analysed for immunoglobulin isotype, 10 were of the IgGI subclass. In competitive binding studies with 7 antibodies to discriminate epitopes, at least 4 distinct binding sites were identified. By Scatchard analysis on 4 of these antibodies, binding constants of 3 were within the range 3.5-5.3 x 10(-9)M. Unusually the 4th antibody appeared to recognise 2 separate antigen sites with binding constants of 2.1 and 7.5 x 10(-9)M. In a preliminary study to compare patterns of reactivity of a selection of the new antibodies with a limited number of sera from smokers and seminoma patients, results indicate their potential for further typing within the placental group of enzymes.
我们描述了一组针对胎盘碱性磷酸酶（PLAP）产生的单克隆抗体（MAb）的制备及初步特性分析。使用不同形式的抗原呈递物，即表达PLAP或PLAP样的全细胞、胎盘膜或纯化的PLAP来免疫BALB/c小鼠。最初通过ELISA针对免疫材料进行筛选，通过免疫染色针对新鲜冷冻胎盘切片进行筛选，并使用酶捕获测定法针对纯化的PLAP进行筛选。最成功的融合是全细胞免疫后的融合，产生了27种抗体，这些抗体均与胎盘型和睾丸型酶发生反应。当针对一系列人类恶性细胞系进行测试时，这些抗体均显示出大致相似的反应模式。进一步的特性分析确定一种抗体8B6与福尔马林固定石蜡包埋的胎盘切片有强烈反应。当针对一系列常规固定的正常和恶性组织切片进行测试时，该抗体也表现良好。在分析的14种抗体的免疫球蛋白同种型中，有10种属于IgGI亚类。在使用7种抗体进行竞争结合研究以区分表位时，至少鉴定出4个不同的结合位点。通过对其中4种抗体进行Scatchard分析，3种抗体的结合常数在3.5 - 5.3×10⁻⁹M范围内。不同寻常的是，第4种抗体似乎识别2个独立的抗原位点，结合常数分别为2.1和7.5×10⁻⁹M。在一项初步研究中，比较了一系列新抗体与来自吸烟者和精原细胞瘤患者的有限数量血清的反应模式，结果表明它们在胎盘酶组内进行进一步分型的潜力。
