Kawakami Y, Battles J K, Kobayashi T, Ennis W, Wang X, Tupesis J P, Marincola F M, Robbins P F, Hearing V J, Gonda M A, Rosenberg S A
Surgery Branch, National Cancer Institute, Bethesda, MD 20892, USA.
J Immunol Methods. 1997 Mar 10;202(1):13-25. doi: 10.1016/s0022-1759(96)00211-6.
Recombinant human MART-1 protein was produced by bacterial and baculoviral-insect cell expression systems. By immunization with bacterial MBP-MART-1 fusion protein or MBP cleaved MART-1 protein, a rabbit polyclonal and two murine monoclonal antibodies specific for MART-1 were produced. These antibodies specifically detected MART-1 in immuno-precipitation, Western blotting, flow cytometric assays and in immunohistochemical analysis of tissue sections. They also stained cytoplasmic components in melanocytes and most melanoma cells in frozen or paraffin embedded tissue sections, indicating that these antibodies may be useful for the diagnosis of melanoma. One of the monoclonal antibodies M2-7 C10 recognized only human MART-1, but the other monoclonal antibody M2-9 E3 recognized both human and murine MART-1. The size of the human MART-1 molecule detected by SDS-PAGE with these antibodies was approximately 18 kDa, suggesting possible posttranslational modifications in the MART-1 protein. Subcellular fractionation studies suggested that MART-1 was present in melanosomes and endoplasmic reticulum, although known melanogenic enzymatic activities were not detected in the MART-1 protein. These reagents may be useful for biological studies on melanocytes and melanoma cells as well as for the development and monitoring of immunotherapy for patients with melanoma.
重组人MART-1蛋白由细菌和杆状病毒-昆虫细胞表达系统产生。通过用细菌MBP-MART-1融合蛋白或经MBP切割的MART-1蛋白进行免疫，制备了一种兔多克隆抗体和两种对MART-1特异的鼠单克隆抗体。这些抗体在免疫沉淀、蛋白质印迹、流式细胞术检测以及组织切片的免疫组织化学分析中能特异性检测到MART-1。它们还能对冷冻或石蜡包埋组织切片中的黑素细胞和大多数黑色素瘤细胞的细胞质成分进行染色，表明这些抗体可能有助于黑色素瘤的诊断。其中一种单克隆抗体M2-7 C10仅识别人类MART-1，而另一种单克隆抗体M2-9 E3既能识别人类MART-1，也能识别鼠类MART-1。用这些抗体通过SDS-PAGE检测到的人类MART-1分子大小约为18 kDa，提示MART-1蛋白可能存在翻译后修饰。亚细胞分级分离研究表明，MART-1存在于黑素小体和内质网中，尽管在MART-1蛋白中未检测到已知的黑素生成酶活性。这些试剂可能有助于对黑素细胞和黑色素瘤细胞进行生物学研究，以及用于黑色素瘤患者免疫治疗的研发和监测。
