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B cell ELISpot- Positive Control


ShawnAbeynaike

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Hi All,

 

I am just starting out with B Cell ELISpots, and just trying to get the assay running with the positive control (Total Ig) at this stage. (This is the first time B Cell ELISpots are being used in my lab, but we have successfully used T Cell ELISpots). I am using PBMCs from patient blood samples 6-9 days post infection. I have tried using the basic protocol provided by Mabtech. I have also tried stimulating with IL-2 and R848 for 48 hours. Finally I have also attempted substituting wash buffers with PBS, PBS/EDTA and PBS/Tween based buffers which I obtained from a different lab which had success with B Cell ELISpots. I am not seeing any spots. 

 

Any suggestions on what steps I should be looking at this point to troubleshoot? 

 

Thanks,

Shawn

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Hey Shawn and welcome to the forum!

 

When you say no spots, is the membrane totally devoid of any spots or are there just very very few ones? A picture would help a lot from a total IgG well. 

 

Also, are you using Mabtech human IgG ELISpot kit? This one: https://www.mabtech.com/products/3850-2a_human-igg-elispotbasic-alp

 

Finally, after incubating the PBMC for 48h in IL-2 and R848, do you check viability of the cells?

 

 

best,

Christian

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Hi Christian,

 

Yes to all the above. The membrane is totally devoid of spots. Yes I am using the kit you linked. I counted cells before and after incubation with IL-2 and R848. I'm running out of ideas as to what could be going wrong. I am not the first one to try it in our lab without success either.

 

Any suggestions would be helpful.

 

Thanks,

Shawn

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This sounds odd. 

 

Could of course be many things but I just thought of something: what substrate are you using for developing the spots?

 

ELISpot absolutely require a precipitating substrate. ELISA substrates wont work. Could this be something?

 

We ofcourse recommend mabtech BCIP/NBT Plus substrate found here: https://www.mabtech.com/products/3650-10_bcip-nbt-plus-substrate-elispot

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Ok, and here are a few more questions and comments:

 

 

1. You say you run the Mabtech protocol that comes with the product. Have you made any modifications to it or are you following exactly?

2. How many cells are you adding into the plate when doing Total IgG after 48h of stimulation R848+IL-2? I ask because the cells numbers should not be to high. If you add 250,000 PBMC/well then you will get a total black out of spots, ie spots will be so many that they become confluent. A totally dark well can look as a negative one since you cannot see any spots.

3. You say you counted the cells before adding them to the plate, but did you then also check for viability?

4. If your wash buffers (i.e PBS) are totally off in terms of pH and then weird things can happen. You said you had switched out the wash buffers so I guess you had thought about this as well but I just want to check. 

5. Please upload some images of the ELISpot wells. Place high resolution well images straight into a powerpoint, write some lines about what it is and attach it. 

6. When you perform the assay do you do the EtOH activation of the ELISpot plate prior to adding the capture antibody overnight? What percentage of EtOH are you using? What plates are you running for this assay, an image would be helpful. 

7. What is the source of the PBMCs you are runnning and have you checked in FACS the percentage of B-cells? When ironing out issues in an assay it is always good if you are 100% sure you can trust the cells and this means using freshly isolated PBMCs. 

8. Finally, you say your lab has been running T-cell elispot in the past, have those always been successful?

 

 

best,

Christian

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1. Yes we initially tried using the Mabtech protocol exactly, and then tried with different buffers but no luck on either front.


2. I did use approximately 230-240,000 PBMCs per well.


3. I didn't specifically check for cell viability other than just a visual assesment. 


6. I intiially tried the MAIPSWU plates with 70% EtOH and of late have been using the MSIP plates with 35% EtOH for activation.


7. I'm using PBMCs from blood samples but I haven't checking using FACS. I will consider doing this.


8. Our T Cells have mostly been successful. We haven't had any issues particularly at this stage of optimization.


I will try and upload some images for you soon if the problem persists.


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Hey thanks for getting back to me! 

 

If you are analyzing 230,000-240,000 PBMC/well when looking at total IgG I think that number is to high. 50,000 or 25,000 is more appropriate. 

 

An image will help a lot because I will instantly be able to see if you have some reaction going on or nothing at all. 

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