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B cell ELISpot on specific antigen pre-coated ELISA plates


Guest Tyler
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Hi!

 

I've been optimizing an antigen specific B cell ELISpot and am having no luck getting antigen specific spots from stimulated MBCs. I have plenty of total IgG+ spots in those wells, but when it comes to the antigen specific wells, no luck unfortunately. We have only found one supplier of our antigen we are using in coating (we've tested many different concentrations for coating)  and it doesn't seem to be working and are wondering if an ELISpot would work using a pre-coated ELISA plate with our antigen of interest since these are more readily available and work for our ELISAs with serum from the same subjects?

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Dear Tyler,

 

ELISpot using ELISA plates is not very good. Plastic is not able to capture as much protein as PVDF membrane, so the quality of spots goes down a lot. I dont think it is a good idea. 

 

However, what you can do is attempt to biotinylate your antigen instead and use in a reversed B-cell ELISpot. I have met other customers that have had no success with coated antigen but achieved great result using biotinylated antigens. 

 

In the revered B-cell ELISpot you use an anti-IgG antibody in the bottom and capture all IgGs secreted by the added B-cells. Then in the detection step you add the biotinylated antigen and finally visualize spots using streptavidin-ALP. The benefit is that you consume very little antigen this way and the quality of spots often go up. Many times you get an increase in spot numbers as well. 

 

You can buy biotinylation kits from companies such as Innovabiosciences. Very easy to use. 

 

https://www.innovabiosciences.com/products/applications/immunoprecipitaion/lightning-link-rapid-biotin/

 

 

Please come back with more questions!

 

/Christian

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Thanks for the speedy response Christian! I have tested the same protein biotinylated and see no spots either unfortunately. I am starting to suspect the antigen we are using may be the issue as I am getting consistent IgG+ wells. I will have to investigate further. 

 

But just to confirm that I understood your response about using a pre-coated ELISA plate, even if it's a working ELISA kit for our same serum samples, there simply won't be as much antigen available for the specific antibodies to bind to?

 

Thanks again!

 

/Tyler

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Dear Tyler,

I was probably to fast in my first response. After coming home I have contemplated the situation some more and realised you could very well be right. Antigen coated ELISA plates could maybe work.

With coated antigen you typically dont need so much protein bound to the bottom of the well. An ELISA plate might be capable of binding enough antigen for the spots to look good. 

By contrast, I am certain a total IgG assay in ELISA plate will look horrible. But coated antigen is different. 

I will tomorrow talk with other people at mabtech and get their input on this situation and I will come back here in the forum. Hold on!

 

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Dear Tyler,

After taking with a few people here at Mabtech I have learned some new things:

1. An ELISA plate has the capacity to bind around 400ng of protein. This includes the walls as well as the bottom. If you only take the bottom of an ELISA plate, we are talking in the neighborhood of 200ng. This might be too little for B-cell ELISpot to work even with coated antigen. It depends on the size an properties of the antigen itself. In essence, you will have to try and see if it works. I initially thought that ELISA plates had binding capacity of 2000ng, but that was not correct. As a result, I am skeptical to this working. 

2. In your first experiments were you got nice total IgG spots but no antigen specific spots, have you considered the possibility that the population of antigen specific B-cells is simply to small? Are there a way to maybe enrich the B-cells? What cell numbers did you use in your experiment and had the cells been activated before for 3 days with R848+recombinant IL-2?

 

best,

Christian

 

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Hi Christian,

1. Thanks for looking into this for me! I'll stick to the PVDF membrane plates!

2. For my antigen specific wells I coated with both 500 000 cells and 180 000 cells. These cells were stimulated for 5 days with PWM, SAC, CpG and IL-10. I was also considering taking a portion of the cells before stimulation and look at ASCs as well after they've rested for an hour after thawing.

 

/Tyler

 

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Hi Tylsan,

If I understand your question, you're trying to optimize and ELISpot to detect IgG-producing cells from samples that have circulating antibody in their serum. You get good total IgG-positive spots but you can't detect Ag-specific IgG when you coat the wells with your antigen, and you have also tried a reverse ELISpot, capturing total secreted IgG and using biotinylated protein for detection. You can detect Ag-specific responses in your patient samples by ELISA (using pre-coated plates and patient serum) but not in the ELISpot, although you're using a different source of antigen. Is this correct? What antigen are you using (peptide or whole protein) and are you using sorted cells or total PBMC? 

It sounds like we first need to identify the source of the problem before we look at the ELISpot technique itself. Because the cells that produce the antibodies that are in serum/plasma are not necessarily readily found in circulation, I would first test whether the antibodies secreted into the sup of your stimulated cells react in the pre-coated ELISA plates. This will tell you if you're having issues with the ELISpot (and components thereof) or if the problem belies in your cells. If you get reactivity in this assay, then we can take steps to optimize your capture ELISpot, but in this case, then the likely culprit is your protein. You should confirm that the biotinylation step is not occluding a major (or main) epitope(s). This is more likely if you're using peptides than whole proteins, but can happen with both- you can also try tagging your antigen genetically. One thing you can try to do is coat an ELISA plate with your biotinylated protein and confirm that you're maintaining the reactivity of the serum. The activation protocol you are using may also not be sufficient enough if the frequency Ag-specific cells in your samples is low. You can read more on this in the following article:

http://www.sciencedirect.com/science/article/pii/S0022175913000653?via%3Dihub

I hope this helps!

Best,

Franco

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Hi Tyler.

Franco already beat me to answering your question. I think its a great idea to test the supernatant of  your stimulated cells in ELISA to see if you have ag-specific ab there to begin with.  ELISA should be sensitive enough to detect low concentrations of these ag-specific abs. 

As a complement to Francos answer about the stimulation protocol, I would recommend using our protocol with 1µg/ml R848 and 10ng/ml rec.IL-2, BUT instead of stimulating them for 3 days, you stimulate them for 5 days. This will give rise to a greater clonal expansion of your B cells compared to what you get after 5 days. I use this protocol when looking at Ag-specific MBC that exist in extremely low frequencies in circulation.  I have attached the results of the small comparison of B-cell stimulation after 3, 5 and 7 days with R848 and IL-2. 

Please let me know if you need these stimulis, I know that there is a guy close to you that works with this protocol.

Best regard

 

Stimulation time.pptx

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