Kang Y H, Carl M, Watson L P, Yaffe L
J Immunol Methods. 1985 Nov 28;84(1-2):177-96. doi: 10.1016/0022-1759(85)90426-0.
Human natural killer (NK) cells have been reported to express various surface antigens. The majority and the most functionally potent NK cells are Leu-11a (NKP-15) positive cells. Only a small number of functional NK cells express Leu-7 (HNK-1) antigen. In the present study, we have established techniques for immunoelectron microscopic identification of NK cells by mouse monoclonal FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies. Ficoll-Hypaque-isolated peripheral blood lymphocytes (PBL) were reacted with the specific antibodies before or after fixation in a 1% glutaraldehyde/1% paraformaldehyde fixative. Prefixation labeling of viable cells with the antibodies was carried out at 4 degrees C or 37 degrees C. Cells prelabeled with anti-Leu-11a antibody were reacted with secondary antibodies either before or after fixation. Anti-Leu-7 antibody was stained directly via an avidin-biotin-peroxidase (ABC) system, anti-Leu-11a antibody was stained indirectly by the ABC immunoperoxidase procedure via a biotinylated anti-mouse IgG secondary antibody or by a 10 nm or 40 nm colloidal gold-labeled anti-mouse IgG antibody. Results indicate that Leu-7 antigen could be localized by incubation with the specific antibody either before or after 20 min fixation; however, Leu-11a antigen was totally abrogated following the same fixation procedure. The Leu-11a antigen was well stained by the methods of prefixation labeling of cells with anti-Leu-11a antibody and incubation with a biotinylated secondary antibody and the ABC system after fixation. With respect to colloidal gold labeling, better results were obtained when cells were reacted with the gold-labeled antibodies immediately after incubation with anti-Leu-11a antibody but before fixation. Ultrastructurally both Leu-7 positive (+) and Leu-11a positive (+) cells shared common ultrastructural features associated with large granular lymphocytes. Using the above described techniques, we found approximately 2-5% Leu-7+ and 9-15% Leu-11a+ cells in the PBL of healthy donors. The overall results suggest that Leu-11a antigen is more sensitive to glutaraldehyde/paraformaldehyde fixation than Leu-7, since it can be localized only by prefixation labeling procedures; the ABC immunoperoxidase procedure is an ideal technique for labeling NK cells for light and electron microscopic enumeration; the immunogold method provides an adequate technique for labeling NK cells which are designated for ultracytochemical studies.
据报道，人类自然杀伤（NK）细胞表达多种表面抗原。大多数且功能最强大的NK细胞是Leu-11a（NKP-15）阳性细胞。只有少数功能性NK细胞表达Leu-7（HNK-1）抗原。在本研究中，我们建立了通过小鼠单克隆异硫氰酸荧光素（FITC）偶联的抗Leu-11a和生物素化抗Leu-7抗体对NK细胞进行免疫电子显微镜鉴定的技术。用Ficoll-Hypaque分离的外周血淋巴细胞（PBL）在1%戊二醛/1%多聚甲醛固定剂中固定之前或之后与特异性抗体反应。在4℃或37℃用抗体对活细胞进行预固定标记。用抗Leu-11a抗体预标记的细胞在固定之前或之后与二抗反应。抗Leu-7抗体通过抗生物素蛋白-生物素-过氧化物酶（ABC）系统直接染色，抗Leu-11a抗体通过生物素化抗小鼠IgG二抗或10nm或40nm胶体金标记的抗小鼠IgG抗体通过ABC免疫过氧化物酶程序间接染色。结果表明，Leu-7抗原在固定20分钟之前或之后与特异性抗体孵育均可定位；然而，相同的固定程序后Leu-11a抗原完全消失。通过用抗Leu-11a抗体对细胞进行预固定标记以及固定后与生物素化二抗和ABC系统孵育的方法，Leu-11a抗原染色良好。关于胶体金标记，在用抗Leu-11a抗体孵育后但在固定之前立即与金标记抗体反应的细胞获得了更好的结果。超微结构上，Leu-7阳性（+）和Leu-11a阳性（+）细胞都具有与大颗粒淋巴细胞相关的共同超微结构特征。使用上述技术，我们在健康供体的PBL中发现约2-5%的Leu-7+细胞和9-15%的Leu-11a+细胞。总体结果表明，Leu-11a抗原比Leu-7对戊二醛/多聚甲醛固定更敏感，因为它只能通过预固定标记程序定位；ABC免疫过氧化物酶程序是用于光镜和电镜计数标记NK细胞的理想技术；免疫金法为标记用于超细胞化学研究的NK细胞提供了一种合适的技术。
