I have to produce a tumor lysate derivate by some tumor cellular lines related to melanoma.
In order to obtain a lysate as immunogenic as possible, I would like to characterize these tumor cell lines. I am evaluating to use some monoclonal purified commercial antibodies directed against: mage1, mage3, melan/mart, gp100, tyrosinase… and use them in indirect flow cytometry. I am going to buy them from specialized company, Abcam, Pierce, etc. Could anyone help me to choose a defined combination of antigens that could assure me a good characterization of my lines in terms of variety and, mostly, immunogenicity? In other words, which antibodies could you suggest me?
Hello Francesca,
if you are referring to immunogenicity as immunogenic for T-cells, then the panel of differentially expressed tumor antigens includes many of the ones you mentioned (you find a listing of peptide antigens in the peptide database of the cancerimmunity.org site). Before you invest a lot of money in your antibodies, I would recommend to analyze your cells on the mRNA level (which would be a lot cheaper). It is generally accepted, that T-cells potentially recognize a cell which is PCR positive, because in any case, it is not the whole protein, but the peptides derived from its degradation that are presented.
A more precise way would be to analyze your lysate for these peptides (by HPLC), but unless your are a specialist in that field, it may be quite difficult given the crude mixture such a lysate is.
Finally, if you have a T-cell clone for standard melanosomal antigens such as Melan A or Meloe or PRAME, you could use lysate from PCR+ tumor cells, pulse it onto DC and ask, whether your T-cells are activated in any way.
(and if you still want to use antibodies, think about Western Blotting rather than flow...)
Hope this helps,
Matthias
Yes, I was referring to T-cell response. Thank you, Matthias, good suggestions. I will check the database you mentioned. I have got a T cell clone positive to melan A and I will surely use it in a elispot assay to test activation by my lysates loaded on T2 cell line. I ought to buy primers...purified antiboies are not so expensive, and, also, I am a fanatic cytoflower. No way to use HPLC. :) Thank you again.
T2 cells are deficient in antigen-processing. So unless your lysate contains the exact peptide epitope which can be loaded onto the MHC complex, the immunogenic peptide is not going to be presented by T2-cells, even if DC may be able to present the antigen after additional processing.
Good luck,
Matthias
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