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ELISpot assays are extremely sensitive and precise assays that require some consideration of component choice and usage as protocols can vary. This section explores some key factors in developing a successful ELISpot assay: from plate chemistry, to layout of controls, to plate handling practices. Please click on one of the topics below for a discussion of ELISpot plate design considerations and best practices.
Choice of Plate (Membrane) – PVDF membrane plates are recommended, over a mixed cellulose ester format due to slightly improved binding of capture Ab and superior performance in spot detection, particularly for fluorescent applications. PVDF has much tighter & more irreversible antibody binding (Figure 8).
The extreme hydrophobicity of the PVDF material33, may necessitate pre-wetting with alcohol prior to addition of the coating Ab. The potential pitfalls of this step are outlined in a later section. Since the mixed cellulose membrane is hydrophilic, ELISpots can be performed without pre-wetting
Comparison of PVDF and HA (MCE) membrane formats for ELISpot. Scanning electron micrographs (SEM) of top surface of membranes. Both membranes are 0.45µm in pore size and have a very high porosity – meaning it has lots of holes and has a sponge like structure. This results in a large surface area proteins – like the first antibody in an ELISpot assay - to bind to. The pores are evenly distributed, which is important to give distinct spots in the ELISpot assay. Even though both membrane types have comparable high protein binding the binding mechanism differs, due to the different polymers:
The HA membrane – a nitrocellulose membrane - was historically first developed and first adopted into ELISpot assays and publications.
The HA membrane is hydrophillic which means you can coat your antibody straight onto the membrane and the antibody will be able to access into the matrix of this membrane. The protein binds to this membrane by means of an electrostatic binding.
The Immobilon-P (IP) membrane was developed later on and has come to be the preferred membrane choice in Western Blots as well as in ELISpot assays. With the IP membrane you have a much larger surface area.
The IP-membrane is a hydrophobic PVDF, that by its one will push off any aqueous solution. So for the antibody to reach into the pore structure and get maximum binding this membrane has to be prewetted with alcohol prior to coating. The antibody then binds by hydrophobic interaction to this membrane.
The IP has a slightly higher protein binding characteristics than HA (**), and there is less chance of losing protein during multiple washes.
The binding characteristics are different for the two membranes. The phobic Immobilon-P membrane provides a better binding mechanism for the ELISpot assay. Nitrocellulose was used first in this assay and this is the reason for its strong presence in the market.
96 well plates optimized for ELISpot assays have unique structural characteristics including:
Low variability rigid sidewalls
Recessed underdrain for low evaporation
Flat membrane for even spot distribution
White and clear housings that are compatible with all imaging systems
Merck’s Multiscreen®HTS plates for ELISpot come with validated Immobilon®-P (PVDF) or mixed cellulose esters (HA) membrane. All are automation compatible and in full compliance with ANSI/SBS 2004 standards.
Negative/Positive Controls – Relevant controls are crucial to measuring Ag-specific responses via ELISpot. Negative controls routinely consist of cells cultured without stimuli, whereas polyclonal T-cell activators are commonly used as positive controls to confirm both cell and assay functionality.
Positive controls include anti-CD3/CD28 Abs, phytohemagglutinin (PHA) and concanavalin A (ConA). These activators induce secretion of many common cytokines including IFNg, IL-2 (Th1), IL-4, IL-5, IL-10 and IL-13 (Th2). Another common control is the commercially available CEF (Cytomegalovirus, Epstein-Barr virus, Influenza virus) peptide pools. These consist of multiple epitopes from each of the three viruses, to which most healthy individuals (~90%) possess CD8-responding T-cells.
Plate organization - Edge effects – The plate is an array of 8 rows with 12 wells in each. Wells at the periphery of the plate (columns 1 and 12, Rows A and H) are in greater direct contact with surrounding environment and thus may differ from interior wells.
Specifically, medium evaporation from peripheral wells in prolonged cultures may impact overall assay performance. Where possible, the use of “media only” wells around the periphery of the true sample wells can minimize this effect. Merck plates & Manifold have been designed/engineered to minimize the edge effect.
Prewetting - Prewetting is not universally applicable to all ELISpots; its requirement is dependent on the inherent hydrophobicity of the capture Ab; therefore, the pre-wetting protocol should be optimized prior to application and there are some best practices. Comparative testing has previously demonstrated that proper ethanol pre-treatment of PVDF-based MultiScreen®HTS plates (25 μL of freshly prepared 35% v/v ethanol followed immediately by water washes) can lead to increased spot number (better sensitivity) and more sharply defined spots (for more accurate quantitation) (Figure 9). Overtreatment with larger volumes of alcohol, longer exposure time, or more concentrated alcohol can lead to trapping of residual liquid between the membrane and underdrain, which may result in poor assay performance or, more critically, well leakage. Leakage associated with alcohol pre-wetting is not a concern when using ELISpot plates lacking an underdrain (e.g. Merck Catalog No. MAIPSWU10); however, this format may suffer from potential media evaporation during extended culturing as well as sterility issues surrounding the exposed base membrane. Another alternative is to use microplates made with hydrophilic membrane such as mixed cellulose ester. It is also important to note that once plates are ethanol-treated, they must be kept wet for the entire assay.
Coating - In traditional ELISAs, binding to the surface occurs via passive adsorption and requires alkaline conditions (0.2 M sodium carbonate/bicarbonate pH>9) to maximize the electrostatic component of the protein:polystyrene interaction. By contrast, coating of PVDF membranes for ELISpot is mediated solely by hydrophobic forces. For this reason, a phosphate-buffered saline (PBS) buffer (pH 7.4) is commonly used.
Membranes also offer significantly greater surface area (300X) for binding than do polystyrene plates39. To ensure performance while maximizing cost efficiency, it is critical to standardize the amount of capture Ab used per well. For optimal performance, we recommend initial titration of both the coating and detection Abs in tandem. Typically, a good starting point for ELISpot coating is 0.5-1 μg Ab per well (5-10 μg/mLin 100 μL); this is 5-10X greater input than for ELISAs. Lower input can result in more diffuse spot morphology as well as reduced spot number.
Both parameters need to be considered when validating new assay protocols, particularly when determining quantitative expression (spot size) or low frequency events, respectively. Merck and Mabtech offer a wide range of fully validated ELISpot Ab pairs (coating and detection) and plates for the assay of human samples as well as other species.
Blocking- Following incubation with capture Ab, plates should be washed extensively, then blocked and equilibrated for 2 hours at 37 °C with the same culture medium (200 μL/well) that will be used during cell stimulation (minus activator).
Once in blocking medium, sealed PVDF plates can be stored overnight at 4 °C. Longer storage can result in protein precipitation and reduced spot resolution. It is important to note that when plates are removed from 4 °C and allowed to reach room temperature, the sealing tape must also be removed to prevent leakage due to gas expansion.