Dear staff,
I am doing HBsAg-specific B cell ELISPOT assay using your Human IgG ELISPOT Basic (3850-2H), and the plate is Millipore MSIPS4510 (PVDF menbrane)(3654-TP-10).
The goal of the experiment is to evaluate the HBsAg-specific B cell response in PBMCs before and after HBV vaccination in humans without HBsAb. I am currently conducting a preliminary experiment, but the HBsAg specific B cell response is not clear (spot is hardly visible), so I would like to discuss the cause of the problem.
The preliminary experiment was conducted by randomly drawing the blood of the researchers to properly establish the protocol, including the concentration of antigen, without comparing PBMCs before and after hepatitis B vaccine vaccination.
When referring to the HBsAg specific B cell ELISPOT assay protocol performed in another laboratory, pre-stimulation was performed for 5 days, not 3 days, so the stimulation was performed for 5 days with 1 ug/mL R848 and 10 ng/mL IL-2, but no spot was observed (figure1).(one subject's PBMC sample, triplicates, Ag concentration 5, 7, 10ug/mL)
I thought that incubation for 5 days might have a negative effect on cell viability, so I performed stimulation for 5 days (figure2) and stimulation for 3 days (figure3) again, but as shown in the figure, several wells were not stained(two subject's PBMC samples, duplicates, 0.4M and 0.2M). Although accurate comparison was difficult, there was no difference between the 3-day culture and 5-day culture, and it was thought that the antigen concentration of 10 ug/mL would be appropriate.
As shown in figures 2 and 3, the problem of not being dyed was thought to be an abnormality in the plate (the manufacturing date is unknown, but it was purchased 4 years ago). The same plate was newly ordered, and PBMC were stimulated for 3 days, and the HBsAg was 10ug/mL, and the response cell was 0.2M, again (figure 4).
The problem of non-staining was no longer found, but the Ag specific B cell response seemed to be indistinct (spots are almost 0-10 levels), so the experiment was performed again by raising the Ag concentration from 10 to 20 ug/mL (figure 5). But again, the spot doesn't seem to be much different.
What is the reason the spot is barely visible?
Looking at the PC, it seems that there is no problem with PBMC or stimulation.
All of the PBMCs used in the preliminary experiment belong to those who have been vaccinated against hepatitis B vaccine and have antibodies.
I am asking for opinions as I am not sure what the problem is. Thank you.
E.J. Jung
