Functional Assessment of Antibody-Secreting B Cells Using ELISpot Assay

When all of the cells have been harvested, seed the subsets into individual wells of a 12-well cell culture plate at a 1 to 10 x 10⁵ cells per milliliter concentration in fresh RPMI medium. Next, activate the B-cells with 5 micrograms of CpG (ODN 2006) per 1 x 10⁶ cells per milliliter per well in the cell culture incubator for five days.

At the end of the activation, add 30 microliters of 35% ethanol in distilled water to each well of the ELISpot plates for 30 seconds. Then, replace the ethanol in each well with 150 microliters of autoclaved double-distilled water, and incubate the plates at room temperature for 5 minutes to remove the residual ethanol. Perform a PBS wash, followed by the addition of 50 microliters of the polyclonal F(ab')2 fragment of anti-human Ig to each well.

Wash the plates two times with PBS, as just demonstrated. Block the non-specific binding in each well with 200 microliters of fresh PBS with BSA for two hours at room temperature. After another PBS wash series, incubate the wells in 100 microliters of fresh RPMI 1640 medium at 37 degrees Celsius. Then, replace the medium in each ELISpot well with the appropriate number of activated B-cells.

Bring the final volume of each well to 150 microliters with fresh RPMI medium. Then, return the ELISpot plate to the cell culture incubator for 8 to 14 hours. At the end of the incubation period, discard the medium, and wash the wells with 200 microliters of PBS plus Tween 20 for five 3-minute washes.

After the last wash, add the appropriate detection antibodies to their corresponding wells, and incubate the plates for two hours in the dark. After another PBS wash series, add 50 microliters of BCIP/NBT substrate to each well for 5 to 15 minutes at room temperature.

When purple spots appear, stop the reaction with 100 microliters of double-distilled water per well, and rinse the plate with running tap water. Then, air-dry the ELISpot membranes protected from light.