Mernaugh Raymond L, Yan Heping, Chen Dong, Edl Jennifer, Hanley Gregory, Pozzi Ambra, Zent Roy
Department of Biochemistry, Vanderbilt Medical Center, Nashville, TN 37232, USA.
J Immunol Methods. 2005 Nov 30;306(1-2):115-27. doi: 10.1016/j.jim.2005.08.006. Epub 2005 Oct 5.
The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.
肾脏高度分支的集合系统在发育过程中由输尿管芽（UB）通过分支形态发生过程产生。这一过程对于肾脏集合管和肾盂的正常发育至关重要，并受到众多蛋白质在空间和时间上表达的严格控制。为了鉴定与成年肾脏高度分化的集合管相比，输尿管芽中差异表达的细胞蛋白，使用了从小鼠早期输尿管芽或成年肾内髓集合管（IMCD）获得的两种细胞群体。在大鼠中进行了消减免疫策略以产生优先与输尿管芽而非IMCD细胞上的抗原反应的单克隆抗体。此外，还描述了抗体打印技术，这是一种用于确定大量单克隆抗体特异性的新型高通量抗体筛选方法。本手稿中概述的方法具有广泛的适用性，因为它们表明可以在大鼠中使用源自小鼠的细胞进行消减免疫。此外，高通量筛选方法应有助于利用消减免疫来鉴定能够区分密切相关细胞类型中表达的蛋白质差异的抗体。
