Hosokawa S, Muramatsu M, Nagaike K
Biosciences Laboratory, Mitsubishi Kasei Corporation, Kanagawa, Japan.
Cancer Res. 1989 Jan 15;49(2):361-6.
Monoclonal antibodies against human alpha-fetoprotein (AFP) were obtained by the hybridoma technique and studied with regard to their reactivities with the human hepatoma cell lines PLC/PRF/5 and KN, and a spontaneously immortalized cell line derived from fetal liver, NuE, all of which synthesize AFP. One of the monoclonal antibodies, 19F12 (IgG2b) became bound to free AFP which was used as the immunogen with an affinity constant of 3.4 X 10(8) M-1. This value was not much higher than those of two other antibodies, 19B1 (IgG1) and 9D12 (IgG2b). However, only antibody 19F12 showed definite reactivity with AFP-producing cells in analysis using flow cytometry. Immunofluorescence microscopy showed that antibody 19F12 detected AFP over the surface of NuE and PLC/PRF/5 cells with a uniform distribution, whereas definite reactivities of antibodies 19B1 and 9D12 to these cells were not detected. These antibodies did not show the specific binding to a non-AFP-producing human lung cancer cell line, PC-9, or to human peripheral blood lymphocytes. The binding ability of 19F12 to hepatoma cells was shown in both viable and fixed cells. Addition of free AFP inhibited the binding of antibody 19F12 to PLC/PRF/5 cells in a concentration-dependent manner. The specific reactivity of 19F12 to human AFP was also confirmed by immunostaining of a tissue section of human cancer proved to be AFP positive with AFP-specific antisera. In two-dimensional polyacrylamide gel electrophoresis of the antigen (from membrane fraction of PLC/PRF/5 cells)-antibody (19F12) complex, spots derived from the antibody and a spot (pI 4.7, Mr 65,000) corresponding in pI and molecular weight to AFP were detected. Western blot analysis showed that material in the membrane fraction of PLC/PRF/5 cells recognized by antibody 19F12 has the same molecular weight as human AFP derived from placenta. In a study of reactivities to PLC/PRF/5 cells treated with various enzymes, the reactivity of this antibody decreased when cells were treated with protease and trypsin and increased when lipase was used. The binding of 19F12 to AFP was not inhibited by concanavalin A. The antibody 19F12 appeared to recognize an epitope that is considered to be part of the peptide area of AFP. These results indicate that the reactivity, the amount of bound antibodies, and the distribution of monoclonal antibodies on antigen-producing cells vary, respectively, even though these antibodies were produced using the same antigen as an immunogen.(ABSTRACT TRUNCATED AT 400 WORDS)
通过杂交瘤技术获得了抗人甲胎蛋白（AFP）的单克隆抗体，并研究了它们与合成AFP的人肝癌细胞系PLC/PRF/5和KN以及源自胎儿肝脏的自发永生化细胞系NuE的反应性。其中一种单克隆抗体19F12（IgG2b）与用作免疫原的游离AFP结合，亲和常数为3.4×10⁸ M⁻¹。该值并不比另外两种抗体19B1（IgG1）和9D12（IgG2b）高多少。然而，在流式细胞术分析中，只有抗体19F12显示出与产生AFP的细胞有明确反应性。免疫荧光显微镜检查表明，抗体19F12在NuE和PLC/PRF/5细胞表面检测到AFP，呈均匀分布，而未检测到抗体19B1和9D12与这些细胞有明确反应性。这些抗体对不产生AFP的人肺癌细胞系PC-9或人外周血淋巴细胞未显示特异性结合。19F12对肝癌细胞的结合能力在活细胞和固定细胞中均有显示。加入游离AFP以浓度依赖方式抑制抗体19F12与PLC/PRF/5细胞结合。用AFP特异性抗血清对经证实为AFP阳性的人癌组织切片进行免疫染色，也证实了19F12对人AFP的特异性反应性。在抗原（来自PLC/PRF/5细胞膜组分）-抗体（19F12）复合物的二维聚丙烯酰胺凝胶电泳中，检测到源自抗体的斑点以及一个等电点和分子量与AFP对应的斑点（等电点4.7，分子量65000）。蛋白质印迹分析表明，抗体19F12识别的PLC/PRF/5细胞膜组分中的物质与源自胎盘的人AFP具有相同分子量。在用各种酶处理的PLC/PRF/5细胞的反应性研究中，当细胞用蛋白酶和胰蛋白酶处理时，该抗体的反应性降低，而用脂肪酶处理时反应性增加。伴刀豆球蛋白A不抑制19F12与AFP的结合。抗体19F12似乎识别一个被认为是AFP肽区一部分的表位。这些结果表明，尽管这些抗体是使用相同抗原作为免疫原产生的，但它们在反应性、结合抗体量以及在抗原产生细胞上的分布分别有所不同。（摘要截短于400字）
