Baumgart Yvonne, Otto Andreas, Schäfer Angelika, Usbeck Elke, Cott Christiane, Schott Astrid, Tornack Maria, Wenzel Anett, Mossie Andualem, Birkenmeier Gerd
Institute of Biochemistry, University of Leipzig, Liebigstrasse 16, 04103 Leipzig, Germany.
Clin Chem. 2005 Jan;51(1):84-92. doi: 10.1373/clinchem.2004.039636. Epub 2004 Nov 4.
Different molecular forms of prostate-specific antigen (PSA) have been used to differentiate between benign prostatic hyperplasia and prostate cancer. Detecting PSA bound to endogenous inhibitors such as alpha(1)-antichymotrypsin (ACT) and alpha(2)-macroglobulin (alpha(2)M) is often difficult because of epitope masking or sensitivity problems. Here we report the characterization of four novel mouse monoclonal antibodies (mabs) obtained by immunization with PSA-alpha(2)M complexes. Their ability to detect free PSA and PSA-inhibitor complexes was shown, and their epitopes were analyzed by phage display technology.
The properties of the mabs were studied by competition and sandwich assays and by Western blotting. Epitope mapping was performed by screening of a phage display peptide library.
All four mabs recognized free PSA, PSA-ACT, and PSA-alpha(2)M complexes, but to various degrees. With different combinations of mabs in competition experiments, antibodies were identified that enhance binding of other mabs to PSA, forming the molecular basis of a very sensitive assay for the detection of PSA and PSA-ACT complexes. Mabs with highest reactivity for PSA-alpha(2)M were selected to establish an immunoassay for that complex. Western blot analysis revealed that all mabs recognized conformational epitopes of PSA. These findings were supported by phage display results demonstrating mimotopes in the PSA molecule.
The results presented here could aid in the further development of clinically relevant assays for PSA and PSA-alpha(2)M complexes.
前列腺特异性抗原（PSA）的不同分子形式已被用于区分良性前列腺增生和前列腺癌。由于表位掩盖或敏感性问题，检测与内源性抑制剂如α(1)-抗糜蛋白酶（ACT）和α(2)-巨球蛋白（α(2)M）结合的PSA往往很困难。在此，我们报告了通过用PSA-α(2)M复合物免疫获得的四种新型小鼠单克隆抗体（mab）的特性。展示了它们检测游离PSA和PSA-抑制剂复合物的能力，并通过噬菌体展示技术分析了它们的表位。
通过竞争和夹心试验以及蛋白质印迹法研究了单克隆抗体的特性。通过筛选噬菌体展示肽库进行表位作图。
所有四种单克隆抗体均能识别游离PSA、PSA-ACT和PSA-α(2)M复合物，但程度不同。在竞争实验中，通过不同单克隆抗体的组合，鉴定出了能增强其他单克隆抗体与PSA结合的抗体，这构成了一种检测PSA和PSA-ACT复合物的非常灵敏的检测方法的分子基础。选择对PSA-α(2)M反应性最高的单克隆抗体来建立针对该复合物的免疫测定。蛋白质印迹分析表明，所有单克隆抗体均能识别PSA的构象表位。噬菌体展示结果表明PSA分子中存在模拟表位，支持了这些发现。
本文所呈现的结果有助于进一步开发与临床相关的PSA和PSA-α(2)M复合物检测方法。
