Dipartimento di Scienze Biomediche, Università degli Studi di Sassari, Sassari, Italy.
J Immunol Methods. 2012 Aug 31;382(1-2):216-9. doi: 10.1016/j.jim.2012.05.023. Epub 2012 Jun 7.
Synthetic peptides are widely used in indirect ELISA to detect and characterize specific antibodies in biological samples. Small peptides are not efficiently immobilized on plastic surfaces by simple adsorption, and the conjugation to carrier proteins with different binding techniques is the method of choice. Common techniques to conjugate peptide antigens to carrier proteins and to subsequently purify such complexes are time consuming, expensive, and occasionally abrogate immunogenicity of peptides. In this report we describe a simple, fast and inexpensive alternative protocol to immobilize synthetic peptides to plastic surfaces for standard ELISA. The technique is based on use of maleimide-activated bovine serum albumin or keyhole limpet hemocyanin as a protein anchor adsorbed on the polystyrene surface of the microtiter plate. Following adsorption of the carrier protein, sulfhydryl-containing peptides are cross-linked with an in-well reaction, allowing their correct orientation and availability to antibody binding, avoiding the time consuming steps needed to purify the hapten-carrier complexes. The immunoreactivity of peptides was tested by using both monoclonal and polyclonal antibodies in standard ELISA assays, and compared with established coating methods.
合成肽广泛用于间接 ELISA 以检测和鉴定生物样本中的特异性抗体。简单吸附不能有效地将小肽固定在塑料表面上，而与不同结合技术的载体蛋白偶联是首选方法。将肽抗原与载体蛋白偶联并随后纯化此类复合物的常见技术耗时、昂贵，并且偶尔会使肽失去免疫原性。在本报告中，我们描述了一种简单、快速且廉价的替代方案，可将合成肽固定在用于标准 ELISA 的塑料表面上。该技术基于使用马来酰亚胺激活的牛血清白蛋白或贻贝血红蛋白作为吸附在微量滴定板聚苯乙烯表面上的蛋白质锚。在载体蛋白吸附后，含巯基的肽在孔内进行交联反应，允许其正确定向并可与抗体结合，避免了纯化半抗原-载体复合物所需的耗时步骤。通过在标准 ELISA 测定中使用单克隆和多克隆抗体测试肽的免疫反应性，并与已建立的涂层方法进行比较。
