Zhu Yusheng, Valdes Roland, Simmons Christine Q, Linder Mark W, Pugia Michael J, Jortani Saeed A
Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA.
Clin Chim Acta. 2006 Sep;371(1-2):71-8. doi: 10.1016/j.cca.2006.02.023. Epub 2006 Mar 6.
Ligand binding is commonly analyzed using various immunoassays that are generally time-consuming and some may require secondary antibodies or gel electrophoresis which are also time-consuming and sometimes subjective. We introduced various examples for a more rapid approach using pre-activated surface chips which are analyzed by surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Specific applications presented in this study include immobilization of antigen, antibody or oligo DNA on pre-activated chips with subsequent identification of the binding antibodies, antigens or DNA binding proteins to demonstrate the universal utility of this novel approach.
BSA-digoxin conjugate (BSA-Dig), anti-digoxin antibody, anti-urinary trypsin inhibitor (uTi) antibody, or a double stranded oligo nucleotide based on the nucleotide sequence between -91 and -10 of the human CYP 450 2E1 promoter were immobilized on the Ciphergen pre-activated surface chips. Anti-digoxin antibody, BSA-digoxin conjugate, uTi, and CYP450 2E1 promoter binding protein were captured on the chip and identified by SELDI-TOF MS.
A protein with 141kDa was identified from anti-digoxin serum using BSA-Dig chips. This binding was competitively inhibited by addition of digoxin. Using anti-digoxin antibody, a peak at approximately 66kDa was detected in the preparation of BSA-Dig. This peak was also inhibited by free digoxin, suggesting BSA-Dig is detected. uTi fragments with approximately 3kDa to approximately 30kDa in the standard and urine samples were captured on the chip by anti-uTi antibody. Finally, we identified a 95-kDa CYP 450 2E1 promoter binding protein in HeLa cells nuclear extracts.
Bioaffinity SELDI-TOF MS is a powerful and versatile approach for analysis of ligands. It eliminates tracer-labeled secondary antibodies and allows for determination of molecular weights of binding proteins and their ligands directly. This approach may also be considered for the detection of enzymes, receptors, or any other specific ligands.
配体结合通常使用各种免疫测定法进行分析，这些方法一般耗时较长，有些可能需要二抗或凝胶电泳，这同样耗时且有时具有主观性。我们介绍了使用预激活表面芯片的更快速方法的各种示例，这些芯片通过表面增强激光解吸/电离飞行时间质谱（SELDI-TOF MS）进行分析。本研究中展示的具体应用包括将抗原、抗体或寡聚DNA固定在预激活芯片上，随后鉴定结合抗体、抗原或DNA结合蛋白，以证明这种新方法的通用性。
将牛血清白蛋白-地高辛偶联物（BSA-Dig）、抗地高辛抗体、抗尿胰蛋白酶抑制剂（uTi）抗体或基于人CYP 450 2E1启动子-91至-10之间核苷酸序列的双链寡核苷酸固定在Ciphergen预激活表面芯片上。抗地高辛抗体、BSA-地高辛偶联物、uTi和CYP450 2E1启动子结合蛋白在芯片上被捕获并通过SELDI-TOF MS进行鉴定。
使用BSA-Dig芯片从抗地高辛血清中鉴定出一种141kDa的蛋白质。加入地高辛可竞争性抑制这种结合。使用抗地高辛抗体，在BSA-Dig制剂中检测到一个约66kDa的峰。该峰也被游离地高辛抑制，表明检测到了BSA-Dig。标准品和尿液样本中约3kDa至约30kDa的uTi片段被抗uTi抗体捕获在芯片上。最后，我们在HeLa细胞核提取物中鉴定出一种95kDa的CYP 450 2E1启动子结合蛋白。
生物亲和SELDI-TOF MS是一种用于配体分析的强大且通用的方法。它消除了示踪标记的二抗，并允许直接测定结合蛋白及其配体的分子量。这种方法也可用于检测酶、受体或任何其他特定配体。
