Bovine ASC elispot

[Guest Analía]

Hi, we are trying to quantify antibody secreting cells and memory B cells from cow PBMCs. We have purchased Mabtech elispot flex (3150-2h). The animals were immunized with a clostridial vaccine, containing recombinant antigens from 2 clostridium. We did a first assay, using these recombinant antigens to sensitize the plates (after activating the plates with ethanol as recommended). PBMCs were criopreserved and viability after thawing was 85%. We did also stimulate cells with R848 1ug/ml for 72h (5x106 cells/ml on tube).

We added the cells to the previosuly coated plates, after washing with PBS, but did not block as it was suggested in the protocol, and incubate o/n.

Then we did all the washing steps with PBS, added the detection antibody, and after final washes we added the TMB. here we had some doubts, since wells rapidly started to stain blue, but we could not detect spots. after 10 min, we start the washing with water. Many washes and the membranes remained blue. But after drying they resulted clear but no spots could be detected.

We are now discussing how to continue. Any suggestions will be very appreciated.

Thanks!

 

[Christian@mabtech.com]

Welcome, I have a few questions/comments:

1. Since you coated with antigen in this B-cell ELISpot, how much antigen/well did you use?

2. Did you include total IgG analysis in a few wells as a postive control?

3. The TMB substrate you used, was it from Mabtech? For ELISpot, the substrate needs to be precipitating substrate, ELISA substrate will not work. 

4. Once you added your cells to the ELISpot plate, which cells number did you use per well? Did you include a tritration of the cells? This is often adventagous when you unsure of the number of antigen specific B-cells you will have. If the response is very high spots will be so many that the entire well becomes confluent with spots. This then will result in something that very much sounds like what you are describing: wells instantly turned color but we saw no spots...

5. Which plate have you used for the experiment? MSIP model from Millipore with PVDF membrane? This is what we recommend. 

[Guest Analía]

Hi Christina, Thanks for your rapid reply.  I will try to answer your comments:

1. We used 25, 10 and 2.5 ug/ml, 100 ul/well of antigen, diluted in PBS 1X (previosuly filtrated through 0,22um). We did an o/n incubation. Plates were incubated inside a humidity chamber, in a 37ºC, 5%CO2 cell incubator.

2. yes, we included a few wells with anti IgG as control, and we added 200.000 and 50.000 cells/well.

3. We used the TMB for ELISPOT from Mabtech (code 3651-10)

4. We used 3 concentration of cells: 2x105, 5 x104 and 2.5x104 cells/well, triplicate.

5. Plates were purchased form Mabtech (3654 - TP-10), and I understand they correspond to MSIPS 4510 Millipore.

 

[Analia]

Sorry, I noticed I was wrong regarding cells seeded on the plates.

For ASC, we used 500.000, 250.000 and 50.000 cells/well.

For memory B cells (that were pre-incubated for 72h with R848) we used 200.000, 50.000 and 25000 cells/well.

Thank you again for your assistance.

 

[Christian@mabtech.com]

Hey, ok so you did titration of your cells. Good. Possible to share images of your plates? Either captured with a mobile phone or better from a ELISpot reader. Add high resolution images into a ppt, write below the images what is what, and upload here. I am in particular interested in seeing the total IgG wells that you have included in your plates. How do these wells look?

In you last reply I get slighly confused when you seperate "ASC" and memory B cells that were pre-incubated. I am slighly confused because in B-cell ELISpot you typically stimulate ALL your isolated cells with r848+IL2 for 72, wash many times and finally seed the ELISpot plate at various cell concentrations. You here add cells to wells coated with anti-IgG capture mab (ie Total IgG control) and to wells where you have coated with your antigen. Is this what you have done?

It is also possible to take out pbmc from the animal and just immediatly add them into the ELISpot plate without pre-activation, but this only works in siutations where you have in vivo activated plasmablasts ciculating in the blood stream. These cells are only present for like 2 weeks following vaccination and will then go to bonemarrow. After this you must use the pre-activation protocol to get the memory B-cells going that constantly are circulating in the blood stream. 

[Analia]

Hi, thanks again for your advice. I do not have here images of the plates, I will try to obtain them and upload them as you suggest. 

We do have PBMCs obtained from animales that had received a booster 14 days before, and we thought we could detect antibody secreting cells directly.  Besides, we have other animals that had previously ( 3-4 months before) received a booster, and these were the ones we used for assessing memory B cells. The Mabtech elispot flex (3150-2h) that we used only said to simulate with R848, so we did not use IL-2, do you think this could be a problem? Besides, which is the suggested concentration of cells for doing this activation? as we could not find this info we decided to use 5x106/ml, on a 15 ml conic tube, for 72 hs, on the cell incubator, but do not know if this is correct? 

Other thing is that the ELISPOT protocol did not mention to block the plate before adding the cells, so we didn't block, and only washed the plates with PBS1x

As said, I will try to get images to upload here asap.

Thanks!!

 

[Christian@mabtech.com]

Hi again Analia,

The pre-activation should be done at a concentration of 1-2 million PBMC per ml. The 5 million per ml you have used is a bit high, but no disaster. Using a 15 ml conic tube is fine but please have the lid abit loose so you allow CO2 to enter the tube and the cells inside of the 37C incubator. 

The IL-2 is only applicable to human and mouse cells. Sorry. Not needed for bovine PBMC. So all good.

In animals that just have been boosted with vaccine it should be possible to take the cells directly, without preactivation, and get spots in ELISpot. However, the circulating plasmablast can disappear from the bloodstream even on day9 in humans so at day14 there can be none to find. How this works in cow is unknown to me. Maybe they leave even earlier.

Finally: coated antigen does not always work in ELISpot. Some antigens simply dont like being coated to membrane plates and the spots never come out looking good, only big and blotchy. Sometimes nothing although this is rare. In these situations one should consider doing the so called reverse B-cell ELISpot protocol where you use biotinylated antigen, added only during the detection step. This often result in better results. 

Nevertheless, the total IgG wells should always work following pre-activation for 72h. So good if you can share those images.

 

[Analia]

Hi Christian. I have attached a file with the images of the ELISPOT plates, that as you will see have no spots at all. 

Hope you have any comments on how to continue with this setup.

Thanks!

Analía.

Bovine ASC elispot 221209.pdf

[Christian@mabtech.com]

The total IgG wells obviously seem darker indicating that something there is reacting. Questions:1

1. For the cells pre-activated with R848 for 72hours, did you wash them three times afterwards before adding them into the ELISpot plate at 200k, 50k and 20k cells/well? I ask because there is so much IgG produced during the pre-activation. If the cells are only washed twice with not so careful aspiration of the supernatant, there can still be so much free IgG in solution left that wells become completely "blacked out". A 3x wash with good removal of supernatant every time is good practice. 

2. You had 85% viability after thawing, did you check the viability of cells after the 72h pre-activation phase? 

/Christian