This was a great talk. Thank you for sharing. I have one question. This might be a silly one, but what if you add only a peptide (for example, IFN-gamma) to the capture antibody-coated plate without cells? Can you still develop spots with detection antibodies and other reagents? What is the difference between adding the target antigen directly to the plate and adding cells that secrete the target antigen, which is then captured by the plate? I think they are the same in terms of binding targeting antigens to the capture antibody, but when the target antigen is only added without cells, there are no spots. Could you please explain the principle behind it?
Thanks for you question! It sounds like an ELISA format may be better suited for your needs. In ELISpot, spots are formed since a high concentration of the analyte (say IFN-gamma) is secreted at the location of the responding cell that is bound by the capture antibody there. If you are adding a sample filled with IFN-gamma, it will be captured all over the plate which won't lead to spot formation. ELISA would work to detect secreted IFN-gamma in solution and give you the concentration of the analyte in your sample.
Hi :) IFNg generally should produce nice responses after 18 hours, but if your sample is expected to contain fewer rare Ag-specific cells, increasing the incubation time up to 48 hours could be beneficial. We recommend that you optimize incubation time before using precious samples.
Hi, With B cell ELISpot, total IgG wells are used as a positive control for the assay. Wells are coated with anti-IgG mAbs. Negative controls could be wells coated with PBS. Cells from the same donor are added to wells coated with the antigen of interest, the positive control wells and negative control wells. Please see our post all about B cell ELISpot ( www.mabtech.com/knowledge-hub/b-cell-profiling-elispot-and-fluorospot ) and don't hesitate to reach out to us via our website if you have more questions!
Excellent presentation, thank you so much!!
Thank you!
I learned a lot from this excellent presentation. Thank you.
Glad it was helpful :)
This was a great talk. Thank you for sharing. I have one question. This might be a silly one, but what if you add only a peptide (for example, IFN-gamma) to the capture antibody-coated plate without cells? Can you still develop spots with detection antibodies and other reagents? What is the difference between adding the target antigen directly to the plate and adding cells that secrete the target antigen, which is then captured by the plate? I think they are the same in terms of binding targeting antigens to the capture antibody, but when the target antigen is only added without cells, there are no spots. Could you please explain the principle behind it?
Thanks for you question! It sounds like an ELISA format may be better suited for your needs. In ELISpot, spots are formed since a high concentration of the analyte (say IFN-gamma) is secreted at the location of the responding cell that is bound by the capture antibody there. If you are adding a sample filled with IFN-gamma, it will be captured all over the plate which won't lead to spot formation. ELISA would work to detect secreted IFN-gamma in solution and give you the concentration of the analyte in your sample.
After adding the cell and stimulus, it says to incubate for 18-48 hours. How long should I incubate for IFN gamma?
Hi :) IFNg generally should produce nice responses after 18 hours, but if your sample is expected to contain fewer rare Ag-specific cells, increasing the incubation time up to 48 hours could be beneficial. We recommend that you optimize incubation time before using precious samples.
Please, I need a lot more of this for my thesis. Thank you
How to set up a control group for B cell ELISPOT test and what is the positive control group
Hi,
With B cell ELISpot, total IgG wells are used as a positive control for the assay. Wells are coated with anti-IgG mAbs. Negative controls could be wells coated with PBS. Cells from the same donor are added to wells coated with the antigen of interest, the positive control wells and negative control wells. Please see our post all about B cell ELISpot ( www.mabtech.com/knowledge-hub/b-cell-profiling-elispot-and-fluorospot ) and don't hesitate to reach out to us via our website if you have more questions!
We have a new webinar on this particular subject: ua-cam.com/video/odYAuLRB0yE/v-deo.htmlsi=-N6ooNXMT0r2dSBu