In the past the characterization of immunotherapeutics in the guinea pig model was limited to measuring humoral immunity. The ELISpot assay overcomes that limitation and it will help researchers to generate more meaningful data in that animal model. The main advantage of this technique is that the cells are obtained from peripheral blood.
No necropsy is required. This allows for the measurement of not just magnitude but also kinetics of T-cell responses in individual guinea pigs during a natural infection or throughout a vaccine regimen. Demonstrating the procedure will be Holly Pugh.
She's a research associate from Inovio's Preclinical R and D Department. To begin add 15 microliters of 35%ethanol to each well of an ELISpot assay plate. After 60 seconds add 150 microliters of PBS to each well.
And invert the plate to stop ethanol pretreatment. Great care should also be exercised when handling the ELISpot assay plates to avoid contamination or damaging the membrane. To wash the plate add 250 microliters of PBS to each well.
After repeating the wash two more times, add 100 microliters of capture anti-guinea pig interferon gamma antibody VE4 to each well. Then incubate the plate for at least 12 hours at 4 degrees Celsius. After the incubation period wash the plates three times.
Add 200 microliters of blocking buffer to each well. Incubate the plate at room temperature for two hours. Then wash the plates three more times.
Prepare conical tubes with 4.5 ml of room temperature density gradient medium. Then mix an equal volume of Hanks'Balanced Salt Solution with the blood. Layer the diluted blood gradually over the density gradient medium, taking care not to disturb the interface.
After this, centrifuge the tubes at 800 x g for 30 minutes without brakes at room temperature. Remove the tube from the centrifuge, taking care not to disturb the layers. After correctly identifying the layers aspirate the complete buffy coat layer to harvest the PBMCs.
Slowly layer the HBSS diluted blood on the density gradient medium. Avoid any disturbance of the tube and turn off the centrifuge brakes. Transfer the cells to a new 15 ml tube.
Then dilute the cells in R10 medium to a total volume of 15 ml. After pelleting the cells, discard the supernatant and resuspend the cells in 15 ml of R10 medium. Pass the cell suspension through a 70 micrometer cell strainer into a new tube.
Count the cells and determine the number of living cells with the Trypan blue exclusion test. Then dilute the cells with R10 medium. First remove the blocking buffer and wash the plates.
For each PBMC sample indicate triplicates of the positive control, negative control, and each experiment-specific stimulant. Dilute the protein peptide pool in R10 medium to three times the desired final concentration. After this add 50 microliters of the peptide R10 medium mixture to the stimulant testing wells of the plate.
Then add 50 microliters of MT peptide formulation in R10 medium to the negative control wells of the plate. Add ConA in R10 medium to the positive control wells of the plate, then add 100 microliters of the PBMC suspension to all of the wells. Place the ELISpot plate onto an even surface in the incubator and avoid disturbing the plate during the incubation period.
After carefully removing the plate from the incubator, invert the plate to remove the supernatant. Then wash the plate three times. Add 100 microliters of diluted biotinylated detection capture anti-guinea pig interferon gamma antibody N-G3 to each well.
Then incubate the plate at room temperature for two hours. Invert the plate to remove the antibody solution and repeat the wash three times. After removing the PBS from the plate, add 100 microliters of ALP conjugated streptavidin diluted in blocking buffer to each well.
Then incubate the plate at room temperature for one hour. Invert the plate to discard the ALP streptavidin solution and wash the plate twice. Invert the plate and blot it against a clean paper towel to remove any excess PBS.
After this wash the plate with 250 microliters of Ultrapure DI water per well. Invert the plate and remove the excess water with a clean paper towel. Next add 100 microliters of BCIP/NBT substrate solution to each well.
Incubate the plate at room temperature for 20 minutes protected from light. Rinse the plate four times with DI water. Then invert and tap the plate to remove excess water.
Finally, remove the plastic drainage from the bottom of the plate and allow the membrane to dry completely. After successful density gradient centrifugation as previously demonstrated, the red viscous liquid at the bottom of the tube should contain most of the red blood cells. The buffy coat layer is between the layer of plasma and the density gradient medium.
Pretreating the wells prior to adding the capture antibody, the assay displayed improved definition along with a reduction in the number of unspecific spots in the negative control wells. Spots are indicative of interferon gamma producing T-cells within the PBMC population. When attempting this technique, it is important to remember to carefully but quickly process the blood.
This will improve cell viability, improve spot formation, and reduce the formation of nonspecific spots. Don't forget that working with blood and BCIP/NBT substrate can be hazardous and precautions such as wearing appropriate PPE should always be taken while performing this procedure. I believe that this protocol allows researchers to study diseases with an important T-cell component such as TB, Ebola, and HSV.
Importantly, it will refine the development of vaccine protocols in the guinea pig model and it will reduce the use of less relevant animal models.
