Th1 and Th2 cytokines in ELISpot

[Guest Frank_K]

Dear Mabtech,

 

If possible, I would like to have your input on one of our upcoming experiments. As part of a larger project, we would like to analyze responses from mouse Th1 and Th2 cells by ELISpot. For Th2, we would like to analyze IL-4, IL-5 and possibly IL-13. Here we are especially interested in IL-4. It is my understanding that this cytokine is easier to analyze with ELISpot compared to ELISA, is this correct? For Th1 cytokines, we plan to analyze IFN-g. Are there any other Th1 cytokines that are suitable to analyze by ELISpot?

 

Also, I have a more technical question. We have a bunch of ELISpot plates called MAIPS4510 in our lab. I could not find these among the plates you recommend in your plate coating guide, but are these ok to use?

 

Thanks a million!

 

Regards,

Frank

 

[Christian@mabtech.com]

Dear Frank, welcome to the Mabtech forum!

 

Yes, you are absolutely correct, antigen-specific IL-4 production is notoriously difficult to detect using ELISA. This is due to relatively few responding cells releasing low amounts of cytokine, combined with the fact that IL-4 is "consumed" by nearby receptor-bearing cells. The use of the ELISpot technique overcomes these problems, since ELISpot detects IL-4 secretion at the site of the secreting cell, and is thereby less susceptible to "consumption" by other cells.

 

When it comes to Th1, IFN-gamma would of course be a very suitable choice, however, another th1 cytokine that could be of interest is TNF-alpha. Mabtech has just recently released kits for mouse TNF-a ELISA and ELISpot based on our in-house developed monoclonal antibodies. In our hands, these systems perform exceptionally well and with mouse splenocytes in ELISpot, TNF-a backgrounds in medium controls tend to be lower compared to what researchers are used to with human PBMCs. We think TNF-a should also be on your list when screening for Th1 responses, especially in mice. If you are a newcomer to ELISpot, you can evaluate this system using our pre-coated PLUS kits. Please take a look on the examples we have on display in our website:

 

https://www.mabtech.com/mouse-tnf-α-kits-and-reagents

 

The MAIPS4510 from Millipore is a similar plate to that of the MAIPSWU10 that Mabtech currently sell in our website (https://www.mabtech.com/products/3654-ep-10_elispot-pvdf-plate-w-o-underdrain). However, there is one big exception! In MAIPS4510, the undertray is made out of soft plastic, whereas it is of hard, rigid plastic in the MAIPSWU10. An image of this hard plastic undertray is attached as a screenshot below from our Youtube tutorial where the plate itself is taken out from the undertray. The plastic undertray belonging to the MAIPS4510 is more prone to leakage problems during EtOH activation as the soft plastic can easily be bent backwards into the activated PVDF membrane. The plastic is actually so soft that just holding the plate firmly from the underside may cause the undertray to "flex" and touch the sensitive membrane after it is activated. Once the leakage has begun it can be difficult to spot it before the plate is incubated overnight with either capture antibody or with cells.

 

By contrast, the hard plastic of the MAIPSWU10 is totally rigid and will never bend backwards. As long as the back of the membrane stays "protected" from any form of physical contact, this plate model is very robust and easy to work with.

 

If your lab have recently purchased a large number of MAIPS4510, I think you should instantly buy 10 plates of MAIPSWU10. Use those plates first, and then reuse the hard plastic undertrays for the rest of your MAIPS4510. Make the swith from soft undertray to hard undertray before you begin with the EtOH activation. In this way, our main objection against the MAIPS4510 is removed

 

 

For more details, please have a look in our Youtube channel where we give you an introduction to the plate models we sell, their strengths/weaknesses and how to properly active them with EtOH prior to antibody coating.