The 𝘌𝘯𝘻𝘺𝘮𝘦𝘓𝘪𝘯𝘬𝘦𝘥 𝘐𝘮𝘮𝘶𝘯𝘰𝘚𝘱𝘰𝘵 (𝘌𝘓𝘐𝘚𝘱𝘰𝘵) 𝘢𝘴𝘴𝘢𝘺 is a highly sensitive immunological technique designed to detect and quantify 𝘪𝘯𝘥𝘪𝘷𝘪𝘥𝘶𝘢𝘭 𝘤𝘦𝘭𝘭𝘴 𝘴𝘦𝘤𝘳𝘦𝘵𝘪𝘯𝘨 𝘴𝘱𝘦𝘤𝘪𝘧𝘪𝘤 𝘤𝘺𝘵𝘰𝘬𝘪𝘯𝘦𝘴, chemokines, or antibodies [1]. Unlike ELISA, which measures bulk cytokine concentration in supernatants, ELISpot captures single-cell resolution. 

𝗣𝗿𝗶𝗻𝗰𝗶𝗽𝗹𝗲 𝗮𝗻𝗱 𝗠𝗲𝗰𝗵𝗮𝗻𝗶𝘀𝗺 
In an ELISpot plate, each well is pre-coated with an antibody specific to the target cytokine (e.g., IFN-γ, IL-2, TNF-α). After adding and incubating immune cells under stimulation, secreted cytokines are trapped by the capture antibodies directly beneath the cell. Following cell removal, a biotinylated detection antibody binds to the captured cytokine, which is later visualized through an enzyme–substrate reaction, forming a colored ´spot´- each spot representing a single secreting cell [2]. 

This quantitative visualization allows calculation of cytokine-secreting cell frequency, typically expressed as 𝘴𝘱𝘰𝘵-𝘧𝘰𝘳𝘮𝘪𝘯𝘨 𝘶𝘯𝘪𝘵𝘴 (𝘚𝘍𝘜) per 10⁶ cells.  

𝗔𝗽𝗽𝗹𝗶𝗰𝗮𝘁𝗶𝗼𝗻𝘀 𝗶𝗻 𝗜𝗺𝗺𝘂𝗻𝗼𝗹𝗼𝗴𝘆 𝗮𝗻𝗱 𝗖𝗔𝗥-𝗧 𝗥𝗲𝘀𝗲𝗮𝗿𝗰𝗵 
ELISpot is a cornerstone assay for evaluating cell-mediated immunity, particularly in vaccine development, autoimmune disease profiling, and tumor immunology [3]. In CAR-T cell research, ELISpot plays a critical role in measuring cytokine release upon antigen recognition, helping to validate CAR-T functionality and monitor off-target or excessive cytokine responses (cytokine release syndrome) [4].  When applied to CAR-M (chimeric antigen receptor macrophages) or engineered Tregs, ELISpot can help assess polarization and the immune-modulatory potential of these advanced therapies. 

𝗪𝗵𝘆 𝗜𝘁 𝗠𝗮𝘁𝘁𝗲𝗿𝘀 
By linking functional immunology to quantitative single-cell analysis, ELISpot bridges the gap between genetic data (qPCR) and protein-level assays (Western blot, ELISA). It is indispensable in understanding immune activation kinetics and fine-tuning therapeutic cell products before clinical translation.   

𝗤𝘂𝗲𝘀𝘁𝗶𝗼𝗻 𝗳𝗼𝗿 𝘁𝗵𝗲 𝗔𝘂𝗱𝗶𝗲𝗻𝗰𝗲  Have you ever optimized an ELISpot plate? What was your biggest challenge – background noise, spot merging, or uneven cytokine detection? 

Stay tuned for 𝗗𝗮𝘆 𝟰𝟬: 𝗙𝘂𝗻𝗱𝗮𝗺𝗲𝗻𝘁𝗮𝗹𝘀 𝗼𝗳 𝗳𝗹𝘂𝗼𝗿𝗲𝘀𝗰𝗲𝗻𝗰𝗲 𝗺𝗶𝗰𝗿𝗼𝘀𝗰𝗼𝗽𝘆 

𝗥𝗲𝗳𝗲𝗿𝗲𝗻𝗰𝗲𝘀 
1. https://doi.org/10.1016/0022-1759(83)90308-3 
2. DOI: 10.1007/978-1-4939-8567-8_2 
3. DOI: 10.1007/s00262-010-0875-4 
4. DOI:10.1038/s41591-019-0549-5 

#Immunology #CellCulture #CARResearch #ELISpot #Cytokines #CellTherapy #Immunotherapy #CARMacrophages #TcellActivation #SingleCellAnalysis