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franco@mabtech.com last won the day on December 18 2017

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About franco@mabtech.com

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    Business Development & Marketing

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  1. Hi Tylsan, If I understand your question, you're trying to optimize and ELISpot to detect IgG-producing cells from samples that have circulating antibody in their serum. You get good total IgG-positive spots but you can't detect Ag-specific IgG when you coat the wells with your antigen, and you have also tried a reverse ELISpot, capturing total secreted IgG and using biotinylated protein for detection. You can detect Ag-specific responses in your patient samples by ELISA (using pre-coated plates and patient serum) but not in the ELISpot, although you're using a different source of antigen. Is this correct? What antigen are you using (peptide or whole protein) and are you using sorted cells or total PBMC? It sounds like we first need to identify the source of the problem before we look at the ELISpot technique itself. Because the cells that produce the antibodies that are in serum/plasma are not necessarily readily found in circulation, I would first test whether the antibodies secreted into the sup of your stimulated cells react in the pre-coated ELISA plates. This will tell you if you're having issues with the ELISpot (and components thereof) or if the problem belies in your cells. If you get reactivity in this assay, then we can take steps to optimize your capture ELISpot, but in this case, then the likely culprit is your protein. You should confirm that the biotinylation step is not occluding a major (or main) epitope(s). This is more likely if you're using peptides than whole proteins, but can happen with both- you can also try tagging your antigen genetically. One thing you can try to do is coat an ELISA plate with your biotinylated protein and confirm that you're maintaining the reactivity of the serum. The activation protocol you are using may also not be sufficient enough if the frequency Ag-specific cells in your samples is low. You can read more on this in the following article: http://www.sciencedirect.com/science/article/pii/S0022175913000653?via%3Dihub I hope this helps! Best, Franco