Reed J C, Tanaka S, Cuddy M, Cho D, Smith J, Kallen R, Saragovi H U, Torigoe T
Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia 19104-6082.
Anal Biochem. 1992 Aug 15;205(1):70-6. doi: 10.1016/0003-2697(92)90580-z.
A strategy is described for production of monoclonal antibodies against recombinant proteins that are produced using the baculovirus expression system and that requires no prior purification of the protein of interest. Crude lysates prepared from cultured Sf9 insect cells infected with recombinant or control baculoviruses are absorbed to nitrocellulose filters and used in a dot-immunobinding assay for screening hybridomas. The monoclonal antibody-producing hybridomas are derived by immunization of mice with a synthetic peptide corresponding to a hydrophilic region in the recombinant protein of interest. By using the baculovirus-produced recombinant protein as the screening antigen and by comparing antibody binding to filters containing control Sf9 lysates, hybridomas are identified that produce monoclonal antibodies with specific reactivity for the recombinant protein of interest and that can then subsequently be used to assist in the large-scale purification of the recombinant protein from baculovirus-infected cells. We applied this method to recombinant 26-kDa human Bcl-2 (B-cell lymphoma/leukemia-2), an integral membrane oncoprotein that regulates programmed cell death ("apoptosis") in hematolymphoid cells through unknown mechanisms. Two mouse monoclonal antibodies were produced that specifically bound the recombinant Bcl-2 baculoprotein in both solution and solid-phase assays.
本文描述了一种生产抗重组蛋白单克隆抗体的策略，该重组蛋白采用杆状病毒表达系统生产，且无需事先纯化目标蛋白。用重组杆状病毒或对照杆状病毒感染培养的Sf9昆虫细胞后制备的粗裂解物，吸附到硝酸纤维素滤膜上，并用于斑点免疫结合试验以筛选杂交瘤。产生单克隆抗体的杂交瘤是通过用与目标重组蛋白的亲水区相对应的合成肽免疫小鼠而获得的。通过使用杆状病毒产生的重组蛋白作为筛选抗原，并比较抗体与含有对照Sf9裂解物的滤膜的结合情况，鉴定出能产生对目标重组蛋白具有特异性反应性的单克隆抗体的杂交瘤，随后可用于协助从杆状病毒感染的细胞中大规模纯化重组蛋白。我们将此方法应用于重组26 kDa人Bcl-2（B细胞淋巴瘤/白血病-2），这是一种整合膜癌蛋白，通过未知机制调节血液淋巴细胞中的程序性细胞死亡（“凋亡”）。产生了两种小鼠单克隆抗体，它们在溶液和固相试验中均能特异性结合重组Bcl-2杆状蛋白。
