Faresjö Maria
School of Health Sciences, Department of Natural Science and Biomedicine, The Biomedical Platform, Jönköping University, SE-551 11, Jönköping, Sweden,
Methods Mol Biol. 2014;1172:3-12. doi: 10.1007/978-1-4939-0928-5_1.
The enzyme-linked immuno-spot (ELISPOT) technique is a sensitive method used for measurement of elusive immune markers in limited-volume samples. By virtue of the exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses), which was not possible before, is now relatively easy. However, development of a method sensitive enough to pinpoint elusive immune markers at the single-cell level is a challenge since there are a number of demands that have to be fulfilled and traps to avoid, achieving a valuable outcome.To optimize the environment for in vitro culture and analysis of immune spots by ELISPOT, a number of criteria have to be fulfilled: processing of sample and perhaps also cryopreservation of cells before analysis and, for the ELISPOT assay, optimal cell culture, positive and negative controls, antigen concentration, and, finally, development and readout of spots.If these criteria are fulfilled for your ELISPOT assay, you will likely have the opportunity to pinpoint elusive immune markers at the single-cell level. This chapter describes the ELISPOT assay for detection of cytokines (e.g., IFN-γ and IL-4), with focus on the main criteria that affect the assay. However, this method could be easily adapted to measure other immune markers in small volumes of biological samples.
酶联免疫斑点（ELISPOT）技术是一种用于在有限体积样本中测量难以捉摸的免疫标志物的灵敏方法。凭借ELISPOT检测的高灵敏度，以前无法进行的稀有细胞群体频率分析（例如抗原特异性反应）现在相对容易实现。然而，开发一种灵敏度足以在单细胞水平精确检测难以捉摸的免疫标志物的方法是一项挑战，因为有许多要求必须满足，也有许多陷阱需要避免，才能取得有价值的结果。为了优化用于通过ELISPOT进行免疫斑点体外培养和分析的环境，必须满足一些标准：样本处理以及可能在分析前对细胞进行冷冻保存，对于ELISPOT检测，还需要优化细胞培养、阳性和阴性对照、抗原浓度，最后是斑点的显影和读数。如果你的ELISPOT检测满足这些标准，你将有可能在单细胞水平精确检测难以捉摸的免疫标志物。本章介绍用于检测细胞因子（例如IFN-γ和IL-4）的ELISPOT检测方法，重点关注影响该检测的主要标准。然而，该方法可以很容易地适用于在少量生物样本中测量其他免疫标志物。
