Simultaneous detection of multiple cytokines in ELISPOT assays.
Living in the era of multiplex detection systems, it appears attractive to develop enzyme-linked immunospot (ELISPOT) assays for the detection of more than one cytokine released by the same cell. However, despite technical simplicity in building such an assay, several factors have to be considered when designing multiplex ELISPOT assays. We have used four capture antibodies (hIFN-gamma, hIL-2, hIL-4, and hTNF-alpha) either in combination or individually to coat polyvinylidene difluoride membrane-backed Millipore 96-well plates. Several cell stimulations were also used, including Concanavalin A, Phorbol Myristate Acetate (PMA) and calcium ionophore (CaI), phytohemagglutinin, CD3e, and lipopolysaccharide. Biotinylated antibodies were used either individually or combined together to detect secreted cytokines. We have found that when plates were coated with all four capture antibodies and captured cytokines were detected using either one detection antibody or all four detection antibodies combined together, fewer spots could be seen when compared with a plate coated with a single capture antibody followed by using its matched detection antibody counterpart. Interestingly, negative interferences between antibodies were less profound when detection antibodies rather than capture antibodies were mixed together.
Palzer
Sarah
S
R&D Systems Inc., Minneapolis, MN, USA.
Bailey
Tanya
T
Hartnett
Chris
C
Grant
Angela
A
Tsang
Monica
M
Kalyuzhny
Alexander E
AE
D017252:CD3 Complex / Q000008:administration & dosage; D003208:Concanavalin A / Q000494:pharmacology; D016207:Cytokines / Q000032:analysis* / Q000096:biosynthesis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000032:analysis / Q000096:biosynthesis; D007376:Interleukin-2 / Q000032:analysis / Q000096:biosynthesis; D015847:Interleukin-4 / Q000032:analysis / Q000096:biosynthesis; D007476:Ionophores / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D010835:Phytohemagglutinins / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013194:Staining and Labeling; D018417:Th1 Cells / Q000187:drug effects / Q000276:immunology; D018418:Th2 Cells / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000096:biosynthesis
D017252:CD3 Complex; C115335:CD3E protein, human; D016207:Cytokines; D007376:Interleukin-2; D007476:Ionophores; D008070:Lipopolysaccharides; D010835:Phytohemagglutinins; D014409:Tumor Necrosis Factor-alpha; D003208:Concanavalin A; D015847:Interleukin-4; D007371:Interferon-gamma