ELISPOT B cell IgG background and no spot

[Guest Calméjane]

Dear Staffs,

I try to perform B cell ELISPOT and plan to detect antigen (drug)-specific IgG or IgM response.

I coated the plate (MultiScreenHTS IP Filter Plate, Millipore) with anti-human IgG mAbs MT91/145 (ref3859-3-250) or anti-human IgM mAbs MT11/12 (3880-3-250), 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).

For IgM I have good results but not for IgG I have a big violet background and I am not abble to detect spot. 

I tried diefferent timing for revelation but it not the problem. I think the concentration of mAb is not good : I used 15µg/mL for coating and 1µg/mL for detection. 

Do you think it's the problem? 

I repeated this experiment 3 times. 

 

Thanks in advance 

[Jahnmatz]

Hi!

Im sorry that you got this problem 3 times in a row. I will help you as much as possible and im sure that we can sort this out!

First, I have some questions:

1. What type of cells did you use? How were they stimulated?

2. What type of controls did you have, how was the results there?

3. What is the difference between well G7 and G8. There seems to be no background in G7, but a lot in G8.  

Best regards

Peter

 

[Guest Calméjane]

Hello, 

thanks for your answer.

1. It's acrivated PBMCs with a cocktail IL2, IL21, CD40 mAb...

2. I use an ISotype control

3. G7 is control POS IgM and G8 control POS IgG so control NEG IgM 

 

Best

[Jahnmatz]

Hi again

Did you use any negative control for IgG? If so what?

It seems that there is something in your wells that gets captured by the coating antibody and is then captured by the detection antibody eventually leading to this background in IgG.

Often, this "something" comes from the cell suspension, either in the media or some wash-step that contained IgG. It should not be the anti-CD40-mAb since this is most likely a mouse antibody, right? And I guess that before you add your cells to the plate, you had washed them a couple of times right?

On your plate, is there a control well without cells but only coating antibody and detection antibody?

 

Best regards

 

Peter