Development of an IFN-γ ELISpot Assay to Assess Varicella-Zoster Virus-specific Cell-mediated Immunity Following Umbilical Cord Blood Transplantation

The overall goal of the following procedure is to detect IFN gamma production at the single cell level to assess the cell mediated immune response directed against varicella zoster virus or VZV. This is accomplished by first coating the wells of a 96 well alley spot plate with IFN gamma capture monoclonal antibody. In the second step, the cells are plated and stimulated with VZV antigen.

Next, the cells are removed and the captured IFN gamma is labeled with a detection antibody, ultimately IFN gamma production and the magnitude and the quality of the cell mediated immune responses directed against BZV can be quantified. The main advantage of this technique over existing methods like PMHC, tremors and intracellular cytokine staining is that interferon gamma. A spot is robust reproducible and informative assay that can be performed on venous blood samples using relatively low tech equipment such as incubators, centrifuges, and dissection microscopes.

To prepare the plates for the procedure begin by adding 20 microliters of 35%ethanol to each well of the 96 well multi-screen IP white alley spot plates to permeable eyes, the PVDF membranes at the bottom of the wells. After one minute, the membranes should become slightly translucent, immediately manually wash the wells three times with 200 microliters of PBS and then coat each well with 100 microliters of purified mouse anti-human IFN gamma capture antibody. Cover the plates with regular plastic wrap and incubate them overnight at four degrees Celsius the next morning, empty the wells and tap them dry.

Then wash the plates five times with 200 microliters of PBS. Next block the wells with 200 microliters of complete media at 37 degrees Celsius after two hours. Wash the plates with PBS as just demonstrated, refilling the wells with fresh PBS after the fifth wash until the cells are plated prior to plating and stimulation.

Resus suspend the human PBMC of interest at a final concentration of two times 10 to the six cells per milliliter and incubate the cells overnight at 37 degrees Celsius and 5%carbon dioxide in a water jacket incubator. The next morning resuspend the PBMC in a final volume of five milliliters of complete media, and then incubate the cells with 10 microliters of genetically engineered endonuclease from SIA Marsens at room temperature. After five minutes, count the cells to confirm at least a 95%viability.

Then spin down the cells and resuspend the pellet in stimulation media to a final concentration of two times 10 to the six cells per milliliter at 37 degrees Celsius until the stimulation mixtures are added to the plate. Next plate, anti CD three to a final concentration of 0.5 micrograms per milliliter in two wells to serve as the positive control wells. Then add 100 microliters of VZV antigen diluted in stimulation media to the appropriate wells.

Now add 100 microliters of PBMC one drop at a time to all of the wells except the negative control wells and incubate the plate for 20 hours at 37 degrees Celsius and 5%carbon dioxide without shaking, moving, or stacking the plates during the incubation. Control the irradiation efficacy by monitoring the absence of visible signs of cytopathic effects after four days in PBMC cultures. After the stimulation, discard the cells and wash the plates 10 times with PBS plus tween.

Then use a multichannel pipette to incubate the PVDF membrane with 100 microliters of biotin conjugated anti IF and gamma monoclonal antibody per well at room temperature after two hours, wash the wells six times with PBS plus tween and incubate the plate covered with 100 microliters of strept in conjugated with alkaline phosphatase per well at room temperature. After one hour, wash the plates three times with PBS plus tween and three times with PBS alone. Then incubate the plate with 100 microliters of substrate solution for five minutes at room temperature.

Now remove the bottom section of the Ellie spot plate and wash both sides of the membrane and the plate with running distilled water. Finally, air dry the plate and membrane and then examine the wells to enumerate the spots. A radiation of viral stocks leads to consistently higher estimates of the frequency of VZV specific IFN gamma producing cells in PBMC samples obtained from healthy donors at all.

Dilution tested possibly the result of the mitigation of cytopathic effects induced by the live attenuated VZV in the cell cultures IFN Gamma, a live spot performed using twofold dilutions of gamma irradiated live attenuated VZV vaccine and PBMC samples from immunocompetent. Children with a documented history of varicella and or a positive serology for VZV indicates statistically significant differences between the median frequencies of IFN gamma producing cells at the one to 200, one to 401 to 800 dilutions of VZV antigen. Depletion of CD four plus cells from the PBMC samples results in a striking reduction in the frequencies of IFN gamma producing cells obtained using one to 101 to 201 to 400 dilution of VZV antigen.

While the depletion of CD eight plus cells does not lead to such a reduction, positive controls are also affected, suggesting that the majority of cells that produce IFN Gamma in response to stimulation with VZV antigens are CD four plus T cells, or CD four plus antigen presenting cells. The absence of which precludes presentation two and activation of IFN gamma producing CD eight plus T cells Once mastered. This technique can be completed in 12 hours if it's done properly.