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Everything posted by akira

  1. Antigen-specific B cell ELISPOT

    Thank you!! I will let you know the result in the near future! Thank you for your support!
  2. Antigen-specific B cell ELISPOT

    Dear Staffs, This is my first time to perform B cell ELISPOT and plan to detect antigen (drug)-specific IgG or IgM response. My plan is to coat the plate (MultiScreenHTS IP Filter Plate, Millipore) with specific antigen, and use anti-human IgG mAbs MT78/145, biotinylated (3850-6-250) or anti-human IgM mAb MT22, biotinylated (3880-6-250), followed by adding Streptavidin-ALP for ELISpot (3310-10-1000). I am able to detect antigen-specific B cells in PBMC with the similar frequency with flu-specific B cells by FACS, although it is very low frequency. 1. I appreciate if you provide the recommended dilution rate of these Abs to begin with. 2. I wonder if I should use antigen-specific B cell (sorted by FACS) or whole B cells in this ELISPOT. I appreciate if you give the advice for it.. Sincerely,
  3. Antigen-specific B cell ELISPOT

    Dear Christian, Thank you for introducing the reverse B-cell ELISpot and the paper, both of which are very exciting. Especially it fit my case very much. That paper describes that coating with 1 microgram/well of anti-human IgG mAb was needed for the reverse B cell ELISpot, but the concentration of biotin-conjugated Antigen (for detection) was not described, although there was the sentence that the amount of antigen needed is reduced in this assay. Could you recommend the amount of biotin-conjugated antigen (per well), although it depends on the case? In the normal ELISPOT, I used 0.5 microgram/ml of anti IgG mAbs (3850-6-250)... Sincerely,
  4. Antigen-specific B cell ELISPOT

    Let me add one thing to the question above. This time, I want to detect the antigen IgG specific B cells spots, not Antigen-IgM spots. Thank you in advance!
  5. Antigen-specific B cell ELISPOT

    Dear Christian and Jahnmatz, Thanks to your great support and advice, I was able to detect the clear image for antigen-specific B cells as attached. I coated the ELISPOT plates with pure antigen (not biotinylated), followed by adding biotin-conjugated IgM, Streptavidin-ALP, BCIP/NBT. Now I need to use biotin-conjugated antigen for coating the plates in a different setting. In this case, how should I arrange the sets of reagent to have the reaction? I appreciate if you can provide the advice or the recommended reagent on that. Sincerely,
  6. Antigen-specific B cell ELISPOT

    Dear Christian, Thank you for your kind support!! Let me contact if I need further supports. Sincerely, Akira
  7. Antigen-specific B cell ELISPOT

    Dear Jahnmatz, Thank you for quick response!! I will follow the recommendation, and please let me contact you again if I need further support. Sincerely,