Dear colleagues, I have the following problem. I need to detect very small quantity antigen specific T- cells (ASC). After vaccination with hepatitis A. For this reason I just can’t use MHC tetramer technology and BD FastImmune™ Antigen-Specific Cytokine Detection. The only possibilities that I have are ELISPOT and ‘Cytokine Secretion Assay- Cell Enrichment and Detection Kit (PE*)’ – MACS. The both are very sensitive but the second also is muiltiparametric. So I can detect ASC on different T population on Cytometer (I mean simultaneous detection of cytokine production and surface markers) I looked at this method very carefully and I found a problem. Is a method representative? I mean the following. The method is based on enrichment of ASC. Before enrichment with a magnet, anti IFN g positive cells are 0.247 % from CD4 population. After enrichment you have 1292 IFN-+ CD4+ T cells per 106 total CD4+ T cells. That is 0.1292 %. So you lose approximately half of the antigen specific cells. See for example: http://www.miltenyibiotec.com/index.php?site=products-cytokine And after that click on Cell Enrichment and Detection Kit (PE*) for IFN G. In case you have very small quantity of ASC and is not possible to detect them before enrichment how can be sure that you can get their exact number after enrichment. See Cell Enrichment and Detection Kit (PE*) for IL4 for example. Thank you in advance for your support. I must decide the right method for detection of my little population. Do you know if there is protein extract (or better- peptide mixture to get T cell response) from Hepatitis A virus? Do you think ELISPOT is better for detection of such a cells? I really would appreciate any help. Stoyan Dimitrov Klinische Forschergruppe Neuroendokrinologie Medizinische Universität Lübeck Ratzeburger Allee 160 Haus 23a 23538 Lübeck Tel.: +49 (451) 500-3644 Fax: +49 (451) 500-3640
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