Kuciel R, Mazurkiewicz A, Ostrowski W S, Stachura J, Steuden I, Szkudlarek J, Radzikowski C
Institute of Medical Biochemistry, Copernicus Academy of Medicine, Kraków, Poland.
Biotechnol Appl Biochem. 1988 Jun;10(3):257-72.
Monoclonal antibody to purified prostatic acid phosphatase from seminal plasma was produced by fusion of spleen cells from immunized mice with the Sp2/O-Ag 14 cell line. This hybridoma-derived antibody, designated MAb-14, was classified as IgG1 immunoglobulin. The apparent affinity constant of phosphatase.MAb-14 complex formation calculated by using the Langmuir isotherm is 2.4 x 10(-8) M. The molecular weight of the complex formed under the condition of antibody excess was found to be 350K, which suggests that 2 molecules of prostatic phosphatase bind to 1 molecule of the antibody. The MAb-14 antibody bound to phosphatase had a negligible effect on the catalytic activity of the enzyme. All isoenzymatic forms of catalytically active prostatic phosphatase, resolved by isoelectric focusing or by chromatofocusing in different pH gradients, reacted with the monoclonal antibody. Several peptides of Mr 25K to 76K and of 13K to 76K were adsorbed from the prostatic tissue extract and from seminal fluid, respectively, on an MAb-14-Sepharose column. The MAb-14 monoclonal antibody was applied to the immunohistochemical investigation of prostatic phosphatase distribution in normal human prostate gland, in nodular hyperplasia, and in adenocarcinoma of the prostate. Immunostaining was observed in prostatic secretory epithelium, within the luminal content of prostatic glands, and in the neighborhood of prostatic cancer cells. Metastatic prostatic carcinoma was also strongly immunoreactive with the antibody. There was no cross-reactivity with leukocytes, kidney, liver, pancreas, spleen, breast, stomach mucosal, and colon tissues.
通过将免疫小鼠的脾细胞与Sp2/O-Ag 14细胞系融合，制备了针对精浆中纯化的前列腺酸性磷酸酶的单克隆抗体。这种源自杂交瘤的抗体，命名为MAb-14，被归类为IgG1免疫球蛋白。利用朗缪尔等温线计算得出的磷酸酶-MAb-14复合物形成的表观亲和常数为2.4×10⁻⁸ M。在抗体过量的条件下形成的复合物分子量为350K，这表明2分子前列腺磷酸酶与1分子抗体结合。与磷酸酶结合的MAb-14抗体对该酶的催化活性影响可忽略不计。通过等电聚焦或在不同pH梯度下进行色谱聚焦分离得到的所有具有催化活性的前列腺磷酸酶同工酶形式，均与单克隆抗体发生反应。分别从前列腺组织提取物和精液中，在MAb-14-琼脂糖柱上吸附了分子量为25K至76K和13K至76K的几种肽段。将MAb-14单克隆抗体应用于正常人类前列腺、结节性增生和前列腺腺癌中前列腺磷酸酶分布的免疫组织化学研究。在前列腺分泌上皮、前列腺腺腔内内容物以及前列腺癌细胞附近观察到免疫染色。转移性前列腺癌也与该抗体发生强烈免疫反应。与白细胞、肾脏、肝脏、胰腺、脾脏、乳腺、胃黏膜和结肠组织无交叉反应。
