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Why anti-CD28 in all T-cell Fluorospot kits but never in ELISpot kits?

Guest Peter2015-10-23T09:47:30Z

Hi Mabtech!

I found the following question and answer in your FAQ section:

21. Why is the addition of anti-CD28 recommended in the FluoroSpot protocol but not in the ELISpot protocol?

In FluoroSpot analysis the possible biological effect of cytokine capture by several coated antibodies has to be taken into consideration. The presence of of e.g. IL-2 capture mAbs may result in reduced activation of T cells. To circumvent the attenuating effect of IL-2 capture, anti-CD28 mAb can be added to provide a co-stimulatory signal to antigen-specific responses by binding to CD28 on T cells. The co-stimulatory effects of anti-CD28 mAb, as well as a possible impact on non-specific spots can be assessed by comparing cells cultured with or without anti-CD28 mAb.

This FAQ answer left me with some questions unanswered. For example, why does the IFNg/Granzyme B Fluorospot kit include an anti-CD28 stimulatory mAb, whereas it is not included in the IL-2 ELISpotPLUS kit? Couldn't the IL-2 capture mAbs in the ELISpot kit also lead to reduced activation of T cells? And is there really any risk for reduced activation of T cells when capturing IFNg and Granzyme B in the mentioned Flurospot kit? Please clarify your rationale further.

Thank you for top-of-the-line products otherwise.

Regards!

Peter

Mattias@mabtech.com2015-10-23T14:22:28Z

Admin

Dear Peter,

Welcome to the Mabtech forum and thank you for your excellent question.

The decision to include anti-CD28 mAb in the FluoroSpot kits was taken after having observed a decreased IFN-g response when simultaneously measuring IL-2, an effect that could be restored by adding anti-CD28 mAbs. Although this effect was primarily seen in FluoroSpot kits including IL-2, the impact of using anti-CD28 antibody may not only depend on what cytokines that are analyzed but also on which cells and stimuli that are used. Since it is difficult for us to know what cells and stimuli people are working with, the anti-CD28 mAb is included in all our FluoroSpot kits intended for T cell analysis.

The use of anti-CD28 is clearly optional and whether or not one should use it may be tested e.g. by comparing the cytokine A response in wells coated with only anti-cytokine A capture mAb or with anti-cytokine A and anti-cytokine B capture mAbs. Correspondingly, cytokine B can be analyzed in wells coated either with only anti-cytokine B mAb or with both mAbs. If the coating with two capture mAbs reduces the response for one of the cytokines, this suggests that capture of the other cytokine has a negative impact. If this is the case, anti-CD28 may be added to see whether it has a positive, compensatory effect on the response. However, capture effects might not only be related to situations where plates are coated with multiple cytokine mAbs but could, as you suggest for IL-2, also have an effect on the same cytokine that is being analyzed. Thus, addition of anti-CD28 may also be tried in single analyte systems (including ELISpot) and may occasionally help to potentiate responses.

Besides compensating for a capture effect, anti-CD28 may also enhance antigen-specific stimulation by serving as a true costimulatory signal. Here, addition of anti-CD28 may substitute for the lack of proper expression of costimulatory molecules (e.g. CD80, CD86) on antigen-presenting cells (APC).

Best regards,

Mattias

_____________________________

Mattias Enoksson, PhD

Scientific Advisor, Marketing & Sales

Guest Peter2015-10-26T08:30:34Z

Thank you for your prompt and comprehensive reply.

I see your point with using anti-CD28 to compensate for capture effects of cytokine A when analysing cytokine B.

However, the Fluorospot assay is very sensitive, and my guess is that you would detect spots in most situations even when said capture effects occur. Given that an in vitro assay is an already non-physiological setting, I don’t really see a problem with the less number of spots. Maybe the anti-CD28 would be necessary if you’re comparing the Fluorospot assay to an ELISpot assay and really want to see similar results. But within a project most researchers would use the same kind of assay and never have that problem. Right?

Well, probably I’m forgetting situations in which the anti-CD28 would be pivotal. Maybe my guess above is wrong? Could the number of spots be so dramatically reduced that you actually need the anti-CD28 to get any results at all?

I agree that it would be good to test the use of anti-CD28 for compensating for capture effects in both Fluorospot and ELISpot, or even to just enhance the possible cytokine output should it be very low in the experimental setup at hand. For this reason, would you mind selling the anti-CD28 mAb separately as a supplementary product as well?

Regards!

Peter

Christian@mabtech.com2015-10-26T12:58:15Z

Admin

Hey there Peter!

In my experience, the inclusion of anti-CD28 in the FluoroSpot assay will never take the response from zero to "significant". In some cases, I have seen 70% reductions in the number of IL-2 spots when anti-CD28 was excluded from the assay, however, most often, anti-CD28 will more than anything make an existing response stronger as the responding cells secrete more cytokine. At the same time, for many antigens I have tried, anti-CD28 makes no difference at all.

As Mattias said earlier, you can of course elect to exclude anti-CD28 based on the exact reasoning that you bring up; the assay is very sensitive and I am already subjecting my cells to a non-physiological setting, i.e. I will only run FluoroSpot and will stick to my protocol w/o the anti-CD28 antibody At the same time, I think it would be good in the beginning of your project to evaluate a couple donors with your antigen, decide upon which cell numbers to use and to see weather anti-CD28 should be included or not. Maybe you see that with anti-CD28 included, your antigen specific response is much easier to evaluate. Instead of having very faint spots, they a clear and distinct. Overall, I would say that this form of co-stimuli is very accepted in the world of immunology.

The anti-CD28 antibody that we include in our human FluoroSpot kits is availble for purchase in our web shop:

https://www.mabtech.com/products/3608-1-50_anti-human-cd28-mab-cd28-purified

We use it a concentration of 100 ng/ml inside the FluoroSpot well.

Guest Peter2015-10-26T15:40:18Z

Hi Christian and Mattias!

Thank you for sharing your experience and insights.

As you recommend, I will evaluate the addition of anti-CD28 stimulation for a couple of donors in the beginning of my next project.

Thank you also for providing me with a link to the anti-CD28 mAb in your webshop. I don't know why I couldn't find it myself.

Take care!