Moran E, Larkin A, Cleary I, Barnes C, Kennedy S M, Kelehan P, Clynes M
National Cell and Tissue Culture Centre, BioResearch Ireland, Dublin City University, Ireland.
J Immunol Methods. 1998 Oct 1;219(1-2):151-9. doi: 10.1016/s0022-1759(98)00134-3.
A study to determine the feasibility of using archival paraffin wax embedded tissue to generate monoclonal antibodies is described. Specifically, monoclonal antibodies were raised to paraffin wax embedded normal human kidney tissue to test the possibility of producing antibodies to such tissue samples prior to attempting generation of antibodies to valuable archival tissue. Multiple sections (10 x 5 microm) were pooled and dewaxed as for immunohistochemical procedures and combined with Freund's adjuvant for immunization of BALB/c mice in vivo. Immunized spleen cells were fused with SP2 myeloma cells and subsequent clones screened on paraffin wax embedded normal human kidney sections, a range of cell lines and normal mouse tissue. Supernatants from 11 wells (from a total of 90 wells screened) showed different staining patterns on sections of paraffin wax embedded kidney. One clone, 1/11C, (isotype IgG1) which exhibited strong staining on all kidney tubules by immunohistochemical studies (glomeruli interstitium and vessels were unstained) and identified a band at 52 kDa on immunoblots of dewaxed kidney tissue (as used for immunogen) was chosen for further characterization. Immunoblotting of five mammalian cell lines showed differential expression of this 52 kDa band (distinct expression on 3/5, weak expression on 2/5 cell lines) whereas, all cell lines displayed a band at 44 kDa and a third band at 70 kDa was observed on 2/5 cell lines. In mouse tissue extracts, the 52 kDa band was identified in kidney tissue only (not in the lung, liver or spleen) with the 44 kDa and 70 kDa bands weakly expressed in all tissues. This preliminary investigation of a novel approach to identifying possible new antigenic markers or producing monoclonal antibodies which react better to known antigens on sections of paraffin wax embedded tissue showed that this method is feasible. The need to have a comprehensive screening system in place and the ability to identify potentially useful clones after the initial screening is paramount due to the relative scarcity of screening material (archival tissue sections) and the tedious nature of the screening method.
本文描述了一项关于确定使用存档石蜡包埋组织生成单克隆抗体可行性的研究。具体而言，针对石蜡包埋的正常人肾组织制备单克隆抗体，以测试在尝试针对珍贵存档组织生成抗体之前，针对此类组织样本产生抗体的可能性。将多个切片（10×5微米）合并，按照免疫组织化学程序进行脱蜡，并与弗氏佐剂混合用于体内免疫BALB/c小鼠。免疫后的脾细胞与SP2骨髓瘤细胞融合，随后的克隆在石蜡包埋的正常人肾切片、一系列细胞系和正常小鼠组织上进行筛选。来自11个孔（总共筛选了90个孔）的上清液在石蜡包埋肾切片上显示出不同的染色模式。通过免疫组织化学研究，一个克隆1/11C（同种型IgG1）在所有肾小管上均表现出强染色（肾小球、间质和血管未染色），并且在脱蜡肾组织（用作免疫原）的免疫印迹上鉴定出一条52 kDa的条带，该克隆被选用于进一步表征。对五种哺乳动物细胞系的免疫印迹显示，该52 kDa条带存在差异表达（在3/5细胞系上有明显表达，在2/5细胞系上表达较弱），而所有细胞系均显示一条44 kDa的条带，并且在2/5细胞系上观察到第三条70 kDa的条带。在小鼠组织提取物中，仅在肾组织中鉴定出52 kDa条带（肺、肝或脾中未鉴定出），44 kDa和70 kDa条带在所有组织中均弱表达。对一种识别可能新抗原标志物或制备对石蜡包埋组织切片上已知抗原反应更好的单克隆抗体的新方法进行的初步研究表明，该方法是可行的。由于筛选材料（存档组织切片）相对稀缺且筛选方法繁琐，因此建立一个全面的筛选系统以及在初始筛选后识别潜在有用克隆的能力至关重要。
