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Simultaneous detection of multiple cytokines in ELISPOT assays
Sarah Palzer1, Tanya Bailey, Chris Hartnett
1R&D Systems Inc., Minneapolis, MN, USA.
Insights
Developing multiplex enzyme-linked immunospot (ELISPOT) assays for detecting multiple cytokines requires careful antibody selection. Combining detection antibodies, rather than capture antibodies, minimizes interference for more accurate results in cytokine detection assays.
Area of Science:
- Immunology
- Biotechnology
- Cellular Biology
Background:
- Multiplex detection systems are increasingly important for analyzing multiple analytes simultaneously.
- Enzyme-linked immunospot (ELISPOT) assays are valuable tools for quantifying cytokine secretion at the single-cell level.
- Developing multiplex ELISPOT assays for simultaneous cytokine detection presents unique challenges.
Purpose of the Study:
- To investigate the feasibility and optimize the design of multiplex ELISPOT assays for detecting multiple cytokines from the same cell.
- To evaluate the impact of combining capture and detection antibodies on assay performance.
- To identify strategies for minimizing interference in multiplex cytokine detection.
Main Methods:
- Utilized polyvinylidene difluoride (PVDF) membrane-backed 96-well plates coated with a combination of four capture antibodies (hIFN-gamma, hIL-2, hIL-4, hTNF-alpha) or single antibodies.
- Employed various cell stimulation methods, including Concanavalin A, Phorbol Myristate Acetate (PMA) with calcium ionophore (CaI), phytohemagglutinin, CD3e, and lipopolysaccharide.
- Detected captured cytokines using individual or combined biotinylated detection antibodies.
Main Results:
- Coating plates with all four capture antibodies resulted in significantly fewer detected spots compared to single-antibody coating.
- Combining all four detection antibodies also reduced spot counts but to a lesser extent than combining capture antibodies.
- Negative interference between antibodies was less pronounced when detection antibodies were combined versus when capture antibodies were combined.
Conclusions:
- Multiplex ELISPOT assay design requires careful consideration of antibody combinations to avoid interference.
- Mixing detection antibodies is a more effective strategy for multiplex cytokine analysis than mixing capture antibodies.
- Optimizing antibody combinations is crucial for achieving reliable and sensitive multiplex cytokine detection using ELISPOT assays.
Abstract:
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.
