Nordström I, Ferrua B
Department of Medical Microbiology and Immunology, University of Göteborg, Sweden.
J Immunol Methods. 1992 Jun 24;150(1-2):199-206. doi: 10.1016/0022-1759(92)90079-9.
Detection of cytokine-producing cells can be accomplished by reverse modifications of the ELISPOT assay using cytokine-specific unconjugated and enzyme-labelled antibodies as solid phase capture system and detecting reagents, respectively. However, in certain situations where the secreted cytokine is produced in minute amounts such as in the case of interleukin-1 (IL-1), the sensitivity of the indicator immunoenzyme system employed may be insufficient to permit detection of the corresponding secreting cells. We have developed a novel immunoenzyme amplification procedure that involves the use of a biotinylated secondary anti-enzyme antibody reagent to enhance the signal provided by the primary enzyme-labelled antibody conjugate. Following addition of enzyme-conjugated avidin, ELISPOT assay wells are developed with a suitable chromogen substrate yielding spots located at the former position of cells secreting the analyte under study. As a model system, the detection of IL-1 beta-secreting cells by human peripheral blood monocytes is described.
细胞因子产生细胞的检测可通过对ELISPOT测定法进行反向改进来实现，分别使用细胞因子特异性未偶联抗体和酶标记抗体作为固相捕获系统和检测试剂。然而，在某些情况下，如白细胞介素-1（IL-1）的情况，分泌的细胞因子产生量极少，所采用的指示免疫酶系统的灵敏度可能不足以检测到相应的分泌细胞。我们开发了一种新型免疫酶扩增程序，该程序涉及使用生物素化的抗酶二抗试剂来增强初级酶标记抗体偶联物提供的信号。加入酶偶联抗生物素蛋白后，用合适的显色底物对ELISPOT测定孔进行显色，在分泌所研究分析物的细胞的先前位置产生斑点。作为一个模型系统，描述了通过人外周血单核细胞检测分泌IL-1β的细胞。
