The enzyme-linked immunospot (ELISpot) assay is a universal method for monitoring immune responses in vitro. ELISpot assay is an effective immunoassay tool due to its high sensitivity. It is used for the ex vivo measurement of cytokine or antibody secreting cells at the single-cell level following activation with a suitable stimulus in vitro. The ELISpot assay allows for the visualization of the secretory product of individually activated or responding cells. Each cell can be detected by the assay as long as a characteristic protein is released and specific high affinity antibodies recognizing the protein are available.

The cells of interest are both cultured and activated on plates immobilised with antibodies that capture the associated target analytes released by the stimulated cells. Following target analyte capture, the presence and distribution of these molecules are detected through a sandwich ELISA. A reporter enzyme is added where the analyte is captured. Each spot that develops in the assay represents a single reactive cell.

As a result, ELISpot assay gives both:

1. 1. Qualitative analysis, by providing the type of immune proteins present.
   2. Quantitative results, by the number of responding cells data.

For 1 litre of 10X PBS, weigh-out:

• 80g NaCl
• 2g KH₂PO₄
• 14.4g Na₂HPO₄ ; 2H₂O.

Add distilled water to 1 litre. Dilute the solution to 1X before use. Check the pH of the 1X solution and adjust to required pH : 7.4 +/- 0.1

For one plate, dilute 50µl of Tween 20 in 100 ml of PBS 1X.

For one plate, dissolve 0.2 g of BSA in 20 ml of PBS 1X.

Reconstitute the lyophilised antibody with 0.55ml of distilled water. Gently mix the solution and wait until all the lyophilised material is back into solution. Please note for 1x96 demo kits, detection antibody is provided in liquid form. If not used within a short period of time, reconstituted Detection Antibody should be aliquoted and stored at -20°C.

In these conditions the reagent is stable for at least one year. For optimal performance prepare the reconstituted antibody dilution immediately prior to use. For one plate, dilute 100 µl of antibody into 10 ml of Dilution Buffer and mix well. To avoid nonspecific background, it is recommended to filter the working solution using a disposable syringe and a 0.2µm filter disc.

For optimal performance, prepare the Streptavidin-AP dilution immediately prior to use. It is recommended to centrifuge the vial for a few seconds to collect all the volume at the bottom. For one plate, dilute 10 μl of Streptavidin-AP conjugate into 10 ml of Dilution Buffer and mix well. Do not keep this solution for further experiments. To avoid nonspecific background, it is recommended to filter the working solution using a disposable syringe and a 0.2µm filter disc.

The reagent is ready-to-use. It should be clear to pale yellow. If precipitates occur, filter the solution using a disposable syringe and a 0.2µm filter disc

Cells can either be stimulated directly in the antibody coated wells (Direct) or, first stimulated in 24 well plates or flask, harvested, and then plated into the coated wells (Indirect). The method used is dependent on 1) the type of cell assayed 2) the expected cell frequency. When a low number of cytokine producing cells are expected it is also advised to test them with the direct method, however, when this number is particularly high it is better to use the indirect ELISpot method. All the method steps following stimulation of the cells are the same whatever the method (direct/indirect) chosen.

We recommend using the following polyclonal activation as a positive control in your assay. We recommend to first pre-stimulate PBMC at 1x10⁶ cells per ml in culture medium (e.g. RPMI 1640 supplemented with 2mM L-glutamine and 10% heat inactivated fetal calf serum) containing anti CD3 antibody (we suggest to use B-B11, ref. 854.010.000, at 1 µg/ml) and IL-2 at 20 ng/ml for 3 days. Then wash the cells once to remove the stimulating antibody and dilute the cells in medium containing IL-2 only for 24 hours.

Harvest cells and resuspend in culture medium added with 5 ng/ml PMA and 500 ng/ ionomycin (Sigma, Saint Louis, MO). Distribute 1x10⁴ to 3x10⁴ cells per 100 µl in required wells of an antibody coated 96-well PVDF plate and incubate for 15-20 hours in an incubator. For other stimulators incubation times may vary, depending on the frequency of cytokine producing cells, and should be optimised in each situation.

Dilute PBMC in culture medium to give an appropriate cell number (same number of unstimulated cells as stimulated sample cells) per 100 µl with no stimulation.

Dilute PBMC in culture medium and stimulator of interest (i.e. Sample, Vaccine, Peptide pool or infected cells) to give an appropriate cell number per 100 µl. Optimal assay performances are observed between 1x10⁵ and 2.5x10⁵ cells per 100 µl. Stimulators and incubation times can be varied depending on the frequency of cytokine producing cells and therefore should be optimised by the testing laboratory.

Note: For optimal performance prepare the Streptavidin-AP dilution immediately prior to use. Note: spots may become sharper after overnight incubation at 4°C in the dark