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akira

Antigen-specific B cell ELISPOT

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Dear Staffs,

This is my first time to perform B cell ELISPOT and plan to detect antigen (drug)-specific IgG or IgM response.

My plan is to coat the plate (MultiScreenHTS IP Filter Plate, Millipore) with specific antigen, and use anti-human IgG mAbs MT78/145, biotinylated (3850-6-250) or anti-human IgM mAb MT22, biotinylated (3880-6-250), followed by adding Streptavidin-ALP for ELISpot (3310-10-1000).

I am able to detect antigen-specific B cells in PBMC with the similar frequency with flu-specific B cells by FACS, although it is very low frequency.

1. I appreciate if you provide the recommended dilution rate of these Abs to begin with.

2. I wonder if I should use antigen-specific B cell (sorted by FACS) or whole B cells in this ELISPOT.

I appreciate if you give the advice for it..

Sincerely,

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Hi Akira.

I could probably help you with this.

1. The detection antibody for IgG (MT78/145) and IgM (MT22) could be used at 0.5ug/ml (1:1000 dilution).

2. If you noticed that the frequency of antigen-specific B-cells were low, I would recommend using non-sorted PBMC (250-500k cells/well). Since the recovery of purification is not 100%, there is a possibility that you loose important cells in the process. If you want to look at resting MBC, I would recommend pre-stimulation of PBMC for 5 days using R848 and rec.IL-2 before use in the ELISpot.

I have attached an article describing this protocol for stimulation (although for FluoroSpot).

Dont hesitate to ask if there is something else.

Good luck!

Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot.pdf

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Guest akira

Dear Jahnmatz,

I want to ask one more.

To  detect total IgM spots, which antibody do you recommend  as a coating antibody? I can't find the "Human IgM mAb Guide" in  the  webpage.

Also I appreciate  if you can tell  me  whether "3880-6-250 anti-human IgM mAb MT22, biotinylated" is compatible as a detection antibody  in  case  of detecting total IgM.

Thank you for your kind support.

 

Sincerely

 

image.png

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Dear Akira,

Sorry we missed you post! 

For Human IgM we recommend the following setup:

Capture mAbs (MT11/12) at 15ug/ml coating concentration (Code 3880-3-1000)

Biotinylated detection mAb (MT22) at 1ug/ml detection (Code 3880-6-1000)

And yes, the MT22 detection antibody works fine in Total IgM detection. 

best,

Christian

 

 

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Dear Christian and Jahnmatz, 

Thanks to your great support and advice, I was able to detect the clear image for antigen-specific B cells as attached.

I coated the ELISPOT  plates with pure antigen (not biotinylated), followed by adding biotin-conjugated IgM,  Streptavidin-ALP, BCIP/NBT.

Now I need to use  biotin-conjugated antigen for coating the plates in a different setting. In this  case, how should I arrange the sets of reagent to have  the reaction?

I appreciate if you can provide the advice or the recommended reagent on that.

Sincerely,

image.png

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Dear Akira, results seem to have worked well with you coated antigen and detection of antigen specifc IgM! Nice!

However, like you point out, in your second experiement using a biotinylated antigen, the first approach will not work since this setup is based on streptavidin-ALP in the detection phase. You will "short-circuit" the assay and end up with totally dark wells if you try it. Its a no go. 

Instead, I would suggest you do the so called reverse B-cell ELISpot! Here you coat the membrane with anti-IgM capture antibodies (MT11/12). You then add your cells containing the B-cells. During incubation all IgM antibodies secreted will get captured. After incubation you wash away the cells using PBS. Then you add your biotinyalated detection antigen for 2h. This will get captured only by the captued IgM specific for your antigen. You wash and visualize these spots using SA-ALP and BCIP/NBT PLus as normal. The results is often better looking and more senstive assay compared to when coating with the antigen itself. 

In this paper they show some comparisons:

https://pubmed.ncbi.nlm.nih.gov/23454005/

 

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Dear Christian,

Thank you for introducing the reverse B-cell ELISpot and the paper, both of which are very exciting. Especially it fit my case very much.

That paper describes that coating with 1 microgram/well of anti-human IgG mAb was needed for the reverse B cell ELISpot, but the concentration of biotin-conjugated Antigen (for detection) was not described, although there was the sentence that the  amount of antigen needed is reduced in this assay.

Could you recommend the amount of biotin-conjugated antigen (per well), although it depends on the case? In the normal ELISPOT, I used 0.5 microgram/ml of anti IgG mAbs (3850-6-250)...

Sincerely,

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