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HBsAg-specific B cell ELISPOT assay

Guest E.J. Jung

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Guest E.J. Jung

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






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Dear E.J. Jung,

Looking at your images I do think you have spots when coating with Hepatitis B antigen. However, the spots are rather faint and not so many. 

Your reader seems to have problem counting these faint spots. Take for example well A4 in figure 4 above. I can with my eyes see that that well contains in the neighbourhood of 30 spots, but you reader is only counting 12. Can you increase the sensitivity of the spot-counting algorithm? 

Also, do I understand correctly you are running 400K and 200K cells/well? Some antigen specific B-cells are very low in frequency. After 3 days in activation you do not only incubate B-cells into the ELISpot plates. Plenty of other cells tag along and only around 10% of fresh PBMC is B-cells. 

When coating antigens to PVDF membrane you risk damaging the epitopes of the antigen. They get smushed up against the membrane structure. By reversing the B-cell ELISpot, utilizing biotinylated antigen during the detection phase and using a coated monoclonal to capture all secreted IgG in the bottom, spots often become much nicer looking and more plentiful. Sometimes you can go from having no spots with coated antigen to clear nice responses with biotinylated antigen. Is this something you can try?

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Hi E.J. Jung

My name is Peter Jahnmatz and I totally agree with Christian, MBC to HBsAg is tricky to find since they are so few in numbers. I have worked with optimisation of a HBsAg MBC assay before. I conducted a study with the similar approach as you were describing. I used the B-cell FluoroSpot assay with peptide-tagged HBsAg-S. Here is a link to that study https://pubmed.ncbi.nlm.nih.gov/31809709/.

If you want to, we could have a trouble shooting session and I can show you the reversed approach and the advantages of it compared to coating.

Please send me an email to peter@mabtech.com if you are interested.

Have a great holiday!  

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  • 2 weeks later...
Guest E.J. Jung

Thank you very much for your valuable comments!
As you commented on, I am considering reverse ELISPOT.
I'd like to ask for additional opinions. Is 5-day culture better than 3-day culture in the pre-stimulation stage? As the incubation time increases, I am concerned that the cell viability may not be good.

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  • 3 months later...

Hi, what culture conditions does Mabtech use for pre-stimulation (cell density and vessel)? I wasn't able to find this information from your papers. Is it also the same for mouse splenocytes? Thank you.

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We recommend doing the pre-incubation with at a cell density of 1million PBMC/ml. The vessel can be a 15ml falcon tube but some people prefer the 5ml falcon tubes because the pellet becomes less "concetrated" with a 5ml tube compared to 15ml. The bottom is more cone-shaped with 15ml tubes. 

It is smart to loosen the top screw abit so that CO2 can get to the cells easily. If the lid is too tight CO2 cannot get in which is bad for the cells. 

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  • 1 month later...
Guest Kirsten


Our lab is running an HBsAg-specific B cell ELISPOT in mouse using the Mouse IgG ELISpot BASIC kit (HRP) (3825-2H), however we often don't see any positivity and can't tell if it is due to the assay or lack of response. What positive control would you recommend for this assay? Thank you!

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Hello Kirsten,

In all B-cell ELISpots I strongly recommend running total IgG analysis as a positive control. So you have some wells coated with your antigen, and then in others wells were you have coated with anti-IgG. 

It is good to go down in cell number for the total IgG control since spot numbers will naturally be much higher compared to any antigen wells. 

If you get spots in total IgG wells you know that the cells are viable and that your ELISpot has worked. If you still then lack spots in you antigen wells it is either due to no antigen specific cells existing or being too few in the well, or that the antigen is not suitable for coating to the membrane. In case of the latter, I recommend trying the reverse B-cell ELISpot approach with biotinylated antigen. 

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