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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

 

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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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