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Antigen-specific IgG spots by antibody-negative donors


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Dear Mabtech staff and researchers,

I am conducting HBV Surface antigen (HBsAg)-specific B cell IgG ELISPOT assay using U-Cytech kit (CT780-PR5). Attached is the image of the result.

This time PBMCs from some healthy vaccinated (i.e. positive for anti-HBs) donors and clinically uninfected donors with no history of vaccination (i.e. negative for serum HBsAg, anti-HBsAg and anti-HBcAg) were stimulated with rIL2 and R848 for five days. PVDF membrane plate (Millipore, MSIPS4510) was coated with four recombinant SHBs antigens, PBS or IgG capture antibodies.

The problem is that some Ab-negative donors show clearly positive spots for HBsAg (the yellow circle). I would appreciate if you could give me any advice on the possible causes of this result and the way to improve this with your products. I also would like to ask if anybody has similar experiences. Thank you.

Keisuke

 

 

ELISPOT20210708.pdf

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Dear Keisuke,

In B-cell ELISpot there is this phenomenon we call sticky Ig. In some donors there are a subset of B-cells secreting Ig that just stick to almost anything. This is most common in IgM and IgA secreting B-cells but it does also happen in IgG. The only way to counteract this is to move over to the reversed B-cell ELISpot protocol instead. This is usually the best approach overall for B-cell ELISpot because you often increase the sensitivity of the assay and you consume less antigen. But it requires you to label your antigens with biotin. On the other hand, sticky Ig phenomon typically show up in the PBS control which is not happening in your assay. But still, I dont think it can be totally striked off. 

Anther scenario could be that this negative donor does in fact have a history of Hepatitis B. Possible? 

 

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Dear Christian,

Thank you for your kind advice.

Dose "reversed B-cell ELISPOT protocol" mean FluoroSpot?
Is it possible with biotinylated antigen and ELISPOT reader?
Even in the reversed protocol, I suspect that the “sticky” IgG trapped on the membrane also will bind to the biotinylated antigens used for detection.

We also thought about the possibility of the occult infection or past infection because the donors are elder people. However, such phenomenon is not rare and we currently think it is unlikely. On the other hand, if the spot is representing truly specific IgG, it is interesting.

Keisuke

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Dear Keisuke,

The great thing about reverse B-cell ELISpot is that the "sticky" Ig phenomenon is pretty much fixed. The biotinylated antigens do not tend to bind randomly to captured IgG. Please note that the reversed B-cell ELISpot approach requires you to coat with anti-IgG in the bottom of the ELISpot plate. We recommend using Mabtech:

- Capture mAbs (MT91/145)

https://www.mabtech.com/products/human-igg-elispot-basic-kit-kit-alp_3850-2a

 

 If you biotinylate your antigens the results can absolutly be read in ELISpot reader. Kits for biotinylating antigens can be purchased commercially. 

 

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