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The Standard ELISpot Assay Steps

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A Note on Membrane-Based ELISpot
Membranes offer vastly improved binding characteristics over standard polystyrene surfaces. While a number of options exist, the majority of ELISpots are currently performed on polyvinylidene fluoride (PVDF) membrane plates.

Key Benefits of PVDF for ELISpot
  • Binding of capture antibody (Ab) is governed by hydrophobic interactions between amino acids such as phenylalanine or leucine and PVDF; this association is much stronger than the electrostatic interactions at nitrocellulose surfaces. 
  • Stronger binding interactions translate to greater Ab density on the membrane’s surface, resulting in better-defined spots. 
  • Because the readout for an ELISpot is “spots/well”, the PVDF membrane’s white color provides the ideal backdrop for spot detection and analysis. 
  • The microplate format further offers greater throughput and is amenable to automation; 
  • More samples, more stimuli, or greater numbers of different cytokines can be assayed simultaneously in neighboring wells.

The Steps

  • Cytokine-specific antibodies are immobilized on membrane-bottomed 96-well plates. 
  • Cells (commonly, total peripheral blood mononuclear cells (PBMCs) or purified subsets) are seeded in the presence or absence of stimulating agents.
  • Over time, activated cells begin to secrete cytokines, which bind to the capture Ab in the immediate vicinity of the expressing cell. 
  • Cells are then washed away and spot detection is accomplished through substrate deposition following either a one-step (enzyme-conjugated, cytokine-specific Ab) or two-step (biotinylated Ab/ Streptavidin-enzyme) antibody binding process. 
  • Once the signals are developed, spot numbers can be tallied manually or through use of image-based spot readers with accompanying analysis software. The frequency and total number of responder cells is determined by comparing the number of spots between stimulated and untreated/control wells.

ELISpot Technique Videos: