The overall goal of this ELISpot methodology is to allow the quantification of Human Antibody-secreting B cells from the blood. This method can help answer key questions in the bio-medical field about the immunology of B cells and vaccine research. The main advantage of this technique is that it allows the detection and measurement of anti-body secreting B cells at the single cell level.
Demonstrating the procedure will be Tsung-Chih Tseng, a graduate student from my laboratory. Immediately after obtaining a 10 ml blood sample from median cubital vein into a 15 ml tube containing potassium supplemented EDTA, invert the tube several times to prevent clot formation. Then add 35 ml of autoclaved red blood cell lysis buffer to the cells for a five minute incubation at room temperature.
When light can be observed through the tube, centrifuge the blood and confirm the presence of a white pellet. Remove the residual lysis buffer with 10 ml's of autoclaved PBS and re-suspend the pellet in 10 ml of RPMI 1640 medium. Plate the cells in a 10 cm culture dish for a 30 minute incubation at 37 degrees celsius and five percent carbon dioxide.
Then gently swirl the culture dish a few times and transfer the floating cells into a plastic 15 ml conical tube. After two washes by centrifugation, re-suspend the pellet at five to 10 times tenth of the six cells per ml concentration in 200 micro liters of cold PBS buffer and add 5 micro liters of biotinylated anti-human anti-body cocktail specific for blood cells per one times tenth of the sixth cells. After a 30 minute incubation on ice, wash the cells in a 10 fold excess volume of sterile PBS and re-suspend the pellet in five micro liters of streptavidin conjugated micro beads per one times tenth of the six cells for a 30 minute incubation on ice.
At the end of the incubation add two ml of PBS buffer to the cells and place the tube onto a magnetic stand for eight minutes at room temperature. When the micro beads have attached to the wall of the tube carefully transfer the supernatant into a new conical tube and add 2 more ml of PBS buffer to the cells. After the second supernatant collection, collect the pooled cells by centrifugation and transfer the cells to a five ml polystyrene tube and fresh PBS buffer at a one times tenth of the seven cells per ml concentration.
After preforming non specific blocking, add one microgram of each selection anti-body per one times tenth of the six cells to the tube with gentle mixing for a 30 minute incubation on ice. During the last five minutes of the incubation add five micro liters of 7-AAD to the cells. Then add two milli liters of fresh PBS to the cells and vortex before centrifuging.
Re-suspend the pellet in fresh sorting buffer at a one to five times tenth of the seven cells per milli liter concentration and filter the cells through a 40 micron cell strainer into a new five milli liter polystyrene tube. Then sort the cells by flow cytometry into three 15 ml tubes containing 5mls of fresh RPI medium to collect the naive B cells, memory B cells and plasmablast plasma cells. When all of the cells have been harvested, seed the subsets into individual wells of a 12 well cell culture plate at a one to ten times tenth of the fifth cells per milli liter concentration in fresh RPMI medium.
Next activate the B cells with five micrograms of CPG ODN 2006 per one times tenth of the sixth cells per milli liter per well in the cell culture incubator for five days. At the end of the activation, add 30 micro liters of 35%ethanol and distilled water to each well of the ELISpot plates for 30 seconds. Then replace the ethanol in each well with 150 micro liters of autoclaved double distilled water and incubate the plates at room temperature for five minutes to remove the residual ethanol.
Perform a PBS wash followed by the addition of 50 micro liters of the polyclonal FAB2 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 micro liters of fresh PBS with BSA for two hours at room temperature.
After another PBS wash series, incubate the wells in 100 micro liters 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 micro liters with fresh RPMI medium then return the ELISpot plate to the cell culture incubator for eight to 14 hours.
At the end of the incubation period, discard the medium and wash the wells with 200 micro liters of PBS plus tween20 for five, three minute washes. After the last wash, add the appropriate detection anti-bodies to their corresponding wells and incubate the plates for two hours in the dark. After another PBS wash series add 50 micro liters of BCIPMBT substrate to each well for five to 15 minutes at room temperature.
When purple spots appear, stop the reaction with 100 micro liters of double distilled water per well and rinse the plate with running tap water. Then air dry the ELISpot membrane's protected from light. In this donor PBMC sample, after red blood cell lysis and adherent cell depletion about 10 percent of the lymphocytes were CD19 positive B cells.
In the B cell compartment about 50 percent of the cells were CD27 negative naive B cells and 50 percent were CD27 expressing memory B cells. CPG treatment of purified human B cells and CD19+CD27+for five days as just demonstrated typically results in the generation of 50 to 200 IgM and 10 to 50 IgG anti-body secreting cells from one times ten to the fourth activated B cells. Once mastered, this ELISpot technique can be completed in three hours if it is performed properly.
Before beginning this procedure, it is important to remember to code the place with appropriate capture re-agents for detecting the anti-body secreting cells. After its development, this technique paved the way for researchers in the field of vaccinology to explore vaccine efficacy in large scale trials and longitudinal follow ups. After watching this video you should have a good understanding of how to purify human B cells from the blood.
Separate naive and memory B cells for their differentiation and perform an ELISpot essay to qualify anti-body secreting cells. Don't forget that working with human blood can be hazardous and that precautions such as wearing gloves should always be taken while performing this procedure.
