Christian@mabtech.com reacted to a post in a topic: Why anti-CD28 in all T-cell Fluorospot kits but never in ELISpot kits?
Mattias@mabtech.com replied to a topic in ELISpotDear 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