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Materials Required

Reagents and Solutions

PBS (Phosphate buffered saline) : Dissolve 8g Sodium chloride,0.2g potassium chloride,1.15 g Disodium hydrogen peroxide and .2g potassium dihydrogen phosphate in 800 ml deionized water and make upto 1000ml.
Wash solution (PBS containing 0.25% (v/v) Tween 20) : mix 1.25 ml Tween 20 with 500 ml of PBS.
Blocking solution: PBS containing 5% (w/v) BSA. : Mix 5 g BSA( bovine serum albumin) in 100 ml PBS.
Dilution buffer (PBS containing 1% (w/v) BSA) : Mix 1g BSA in 100 ml PBS.
Complete RPMI- 10 medium: RPMI 1640 medium containing: 10%FBS, heat-inactivated 1 hr at 560C,,2 mM L-glutamine,,50 µM 2-ME (2-mercaptoethanol), 100 µg/ml streptomycin sulfate

Coat nitrocellulose-backed microtiter plate with cytokine-specific primary antibody : Add an optimal amount of purified cytokine-specific primary antibody in coating buffer in a volume of 50 µl( 15µg/ml) to wells of nitrocellulose-backed microtiter plate.

Discard coating antibody. Rinse plate three times with wash solution (200 µl per rinse).

Shake excess liquid from plate and pat bottom of plate with dry absorbent paper.

Discard blocking solution, wash plate three times with wash solution (200 μl per wash).

Shake excess liquid from plate, and pat bottom of plate with dry absorbent paper.

Add 100µl mitogens, antigens, or other stimulus to the cells eg : PHA to 5 µg/ml. Maintain unstimulated cells(medium alone) to be used to assess background cytokine production and in vivo activation.

Incubate cells 6 to 24 hr on a level surface in a humidified 37°C, 5% CO2 incubator.

Wash plate extensively with wash solution (200 μl per wash), shake excess liquid from plate, and pat dry.

Add 50 μl labeled cytokine-specific secondary antibody in ELISPOT dilution buffer to each well. Incubate 2 hr at room temperature.

Discard the antibody and wash plate six times with wash solution (200 μl per wash).

Add 50 μl alkaline phosphatase–labeled detecting antibody or protein and incubate 2 hr at room temperature.

Add 50 μl BCIP solution to each well and incubate 5 to 30 min at room temperature until color (blue spots) develops.

The optimal concentration of cytokine-specific primary antibody should be determined in advance It is generally necessary to use concentrations 5- to 10-fold higher than those used in conventional ELISA assays (e.g., 5 to 10 µg/ml).The antibodies are usually diluted in PBS (pH 7.2 to 7.4) or borate buffer (pH 8.4). Because each antibody used for coating has different binding characteristics, it is necessary to determine the optimal coating conditions for each antibody.

The appropriate concentration of secondary cytokine-specific antibody must be determined in advance (see suggested concentrations) and has to be diluted in PBS containing 1% (w/v) BSA.

For stimulus of the incubated cells mitogens ,antigens or other stimulus is added . Stimulation can occur prior to addition of the cells or during incubation on the plate. For standard ELISPOT analyses, the stimuli can be added directly to the cells while in the nitrocellulose-backed microtiter plates. Also a protein inhibitor such as cycloheximide (100 g/ml) can be used as an additional control. Cycloheximide should be added to 3 or 4 replicate wells, for both unstimulated and stimulated cells, at the initiation of culture.

The cells are incubated for 6 to 24 hr on a level surface in a humidified 37°C, 5% CO2 incubator. The length of incubation depends upon the type of cell involved and the nature of the stimulus. For many cytokines, there is little spontaneous production; thus for the majority of cytokines, a stimulus will be necessary to enumerate cells capable of secreting a given cytokine. Each cytokine has its own specific kinetics, and appropriate conditions must be determined for a given set of stimuli. If the incubation is too short, reduced numbers of spots will result. If the incubation is too long, there will be an increase in the assay background.

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