Abbott Christina, Huang Guo, Ellison Aaron R, Chen Ching, Arora Taruna, Szilvassy Stephen J, Wei Ping
Department of Protein Sciences, Amgen Inc., Thousand Oaks, California, USA.
Hybridoma (Larchmt). 2010 Apr;29(2):103-13. doi: 10.1089/hyb.2009.0095.
Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.
利用杂交瘤技术制备了针对血小板生成素（TPO）同源受体人c-Mpl的小鼠单克隆抗体（MAb），并通过各种检测方法对其进行表征，以证明其特异性和亲和力。确定两种这样的单克隆抗体1.6和1.75在流式细胞术研究中表现优异，对可溶性人c-Mpl蛋白具有两位数皮摩尔（pM）亲和力。两种单克隆抗体均特异性结合经基因工程改造以过表达人c-Mpl蛋白的细胞、表达内源性c-Mpl的永生化人造血细胞系、原代人骨髓和外周血来源的CD34(+)细胞以及纯化的人血小板。在不表达c-Mpl的细胞系上未检测到结合。受体竞争和siRNA敲低研究进一步证实了抗体1.6和1.75对人c-Mpl的特异性。与这些新产生的单克隆抗体形成对比的是，所测试的八种市售抗c-Mpl抗体均未被发现与人c-Mpl特异性结合，因此显示不适合用于流式细胞术研究。因此，单克隆抗体1.6和1.75将是检测细胞表面c-Mpl表达的有用流式细胞术试剂。
