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ELISPOT Quick Guide

Enzyme-linked ImmunoSPOT

ELISPOT assays were originally developed to enumerate B cells secreting antigen-specific antibodies, but subsequently the assay has been adapted for identification and enumeration of cytokine-producing cells at the single cell level. The method employs the sandwich assay approach of the enzyme-linked immunosorbent assay (ELISA), with some variations. The ELISPOT capture antibody is coated aseptically onto a polyvinylidene difluoride (PVDF)-backed microwell plate. The plate is blocked with serum proteins, cells of interest are plated out at varying densities, along with antigen or mitogen, and plates are incubated at 37°C. Cytokine secreted by activated cells is captured locally by the coated antibody on the high surface area PVDF membrane. A second antibody (biotinylated) reactive with a distinct epitope of the target cytokine is employed to detect the captured cytokine. The detected cytokine is then visualized using avidin-HRP, and a precipitating substrate (e.g., AEC). The colored end product (spot) represents an individual cytokine-producing cell. The spots can be counted manually (e.g., with a dissecting microscope) or using an automated reader to capture the microwell images and to analyze spot number and size.

The ELISPOT assay enables analysis of activated or responding cells at the single cell level. By virtue of exquisite sensitivity of the ELISPOT assay, frequency analysis of rare cell populations (e.g., antigen-specific responses) which were not possible by bulk assay methods are now possible. Limits of detection are below 1/100,000.

Mouse ELISPOT Ready-Set-Go! Sets & Antibody Selection Quick Guide
Mouse CytokineELISPOT RSG Set Cat. No.Capture mAbCat. No.Detection mAbCat. No.
IFN gamma 88-7384 AN-18 16-7313 R4-6A2 13-7312
IL-1 alpha 88-7813 ALF-161 16-7011 Rabbit Polyclonal 13-7111
IL-2 88-7824 JES6-1A12 16-7022 JES6-5H4 13-7021
IL-4 88-7844 11B11 16-7041 BVD6-24G2 13-7042
IL-5 88-7825 TRFK5 16-7052 TRFK4 13-7051
IL-6 88-7864 MP5-20F3 16-7061 MP5-32C11 13-7062
IL-10 88-7804 JES5-16E3 16-7101 JES5-2A5 13-7102
IL-13 88-7834 eBio13a 16-7133 eBio1316H 13-7135
IL-17A 88-7370 eBio17CK15A5 16-7175 eBio17B7 13-7177
TNF alpha 88-7328 1F3F3D4 16-7325 mAb Cocktail (XT3/XT22) 13-7326

 
Human ELISPOT Ready-Set-Go! Sets & Antibody Selection Quick Guide
Human CytokineELISPOT RSG Set Cat. No.Capture mAbCat. No.Detection mAbCat. No.
IFN gamma 88-7386
MD-1
16-7317
4S.B3
13-7319
IL-1 beta
88-7810
CRM56
16-7018
CRM57
13-7016
IL-4 88-7849
8D4-8

MP4-25D2
13-7048
IL-5 88-7859
TRFK5
16-7052
JES1-5A10
13-7059
IL-6 88-7869
MQ2-13A5
16-7069
MQ2-39C3
13-7068
IL-10 88-7805
JES3-9D7
16-7108
JES3-12G8
13-7109
IL-12 p70
88-7879
B-T21

C8.6
13-7129
IL-13 88-7839
PVM13-1
16-7139
Rabbit Polyclonal
13-7138
IL-17A 88-7876
eBio64CAP17
16-7178
eBio64DEC17
13-7179
TNF alpha 88-7398
MAb1
16-7348
MAb11
13-7349