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  2. Spots in positive control and not antigen specific

    Agree with Peter. But please also add some well images from the AID reader. Add them into a powerpoint and write in what is what. Then upload here. Will help a lot.
  3. Hi Eunice I will gladly help you with you. However, I think that I need some more information about you assay first. As I understand it you are seeing antigen-specific IgG and IgM spots in ELISpot, but not in FluoroSpot when you run a dual IgG/IgM assay? Is that right? Since you see IgG and IgM spots in the total wells, there seems to be no problem with the detection antibodies or the cells. Please provide some more info on the protocols (you dont have to share specifics, only the general protocol that you use). Best regards Peter
  4. I am Using the AID ELISpot reading machine
  5. Just running a dual fluorospot IgG/IgM and can confirm spots using ELISpot but when I run Fluorospot for the same, I only have responses in the total IgG/IgM wells Any help please
  6. No spots for IFN-gamma

    Ah, very good that we found the issue! Mabtech TMB substrate for ELISpot can be found here: https://www.mabtech.com/products/tmb-substrate-elispot_3651-10 Your strategy for the number of cells to incubate per well sound very reasonable.
  7. No spots for IFN-gamma

    I see! Thats why I am confused as both of them are TMB substrates. I think that is the problem. I had one time had some yellow spots on the membrane with TMB elisa substrate after drying. Thank you! I will try again and update you after acquiring the correct substrate. The cells were seeded such that 1000, 100, 10 and 1 spot is expected, so with 10% transfection efficiency i seed from 10000 to 10 cells.
  8. IL-2 specific activity

    Hi! The IL-2 supplied in the ELISpot kit is used for stimulation of memory B cells in combination with R848. The activity of the IL-2 is confirmed to give more than 500 spots per 50 000 PBMC (per well) with pre-stimulation using 10 ng of IL-2/ml and 1 ug/ml of R848. The biological activity of IL-2 has not been compared with an international reference reagent to establish the number of units. Best regards! Peter
  9. No spots for IFN-gamma

    Hold on, you are running an ELISpot basic kit! What substrate are you using? We have had experience before with people getting zero spots after trying to develop them using ELISA substrate. Will NOT work. The substrate need to be "precipitating" substrate, like BCIP/NBT Plus: https://www.mabtech.com/products/bcip-nbt-plus-substrate-elispot_3650-10
  10. No spots for IFN-gamma

    Dear Kenneth, in all liklihood your transfected cells are not secreting the IFNg cytokine. How many cells are you incubating per well? We occasionally run transfected cells in ELISpot when looking at exotic animal IFNg like Salmon for example. We get massive amount of secretion so we have to keep incubation very short. Only about 2-4h. Rather few cells per well also, around 500-1000. Otherwise the whole membrane is full of spots.
  11. IL-2 specific activity

    Hello, Would you know the specific activity in IU/ug of the human IL-2 provided in your Fluorospot kit (IgG, IgA, IgM fluorospot)? Thanks
  12. No spots for IFN-gamma

    Also, I wish to ask does flag tag mask the monoclonal antibody? And moreover i want to know how your plus and pro plates works. Is it shipped dry for the membrane and pre-coated? So do I need to use ethanol to activate it before I put my cells in for incubation?
  13. No spots for IFN-gamma

    Hi, I am getting zero spots and low background in the human IFNG elispot assay. I am just trying out elispot assay before moving to other applications with human IFN-gamma protein. So I am using 293F (suspension type 293 derivative, serum free) and transfect a CMV driven flag tagged IFNG from geneEZ ORF plasmid, co-transfect with GFP reporter. https://www.genscript.com/gene/homo-sapiens/3458/ifng.html#NM_000619.3 I am using human elispot basic kit with all condition same as suggested protocol on website. But there is no spot for many times i tried, with different cell lines. The reagents are new so I did not add IFNG protein as positive control. We don't have PBMC in the lab. There is zero background, which is quite white.
  14. Antigen-specific B cell ELISPOT

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

    The concentration for the biotinylated antigen should be optimized in trial experiments through a titration. I would recommend something like: 0,1ug/ml 0,5ug/ml 1,0 ug/ml Good luck Akira! Christian
  16. 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,
  17. Antigen-specific B cell ELISPOT

    Dear Akira, results seem to have worked well with you coated antigen and detection of antigen specifc IgM! Nice! However, like you point out, in your second experiement using a biotinylated antigen, the first approach will not work since this setup is based on streptavidin-ALP in the detection phase. You will "short-circuit" the assay and end up with totally dark wells if you try it. Its a no go. Instead, I would suggest you do the so called reverse B-cell ELISpot! Here you coat the membrane with anti-IgM capture antibodies (MT11/12). You then add your cells containing the B-cells. During incubation all IgM antibodies secreted will get captured. After incubation you wash away the cells using PBS. Then you add your biotinyalated detection antigen for 2h. This will get captured only by the captued IgM specific for your antigen. You wash and visualize these spots using SA-ALP and BCIP/NBT PLus as normal. The results is often better looking and more senstive assay compared to when coating with the antigen itself. In this paper they show some comparisons: https://pubmed.ncbi.nlm.nih.gov/23454005/
  18. 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!
  19. 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,
  20. ELISPOT B cell IgG background and no spot

    Hi again Did you use any negative control for IgG? If so what? It seems that there is something in your wells that gets captured by the coating antibody and is then captured by the detection antibody eventually leading to this background in IgG. Often, this "something" comes from the cell suspension, either in the media or some wash-step that contained IgG. It should not be the anti-CD40-mAb since this is most likely a mouse antibody, right? And I guess that before you add your cells to the plate, you had washed them a couple of times right? On your plate, is there a control well without cells but only coating antibody and detection antibody? Best regards Peter
  21. Hello, thanks for your answer. 1. It's acrivated PBMCs with a cocktail IL2, IL21, CD40 mAb... 2. I use an ISotype control 3. G7 is control POS IgM and G8 control POS IgG so control NEG IgM Best
  22. ELISPOT B cell IgG background and no spot

    Hi! Im sorry that you got this problem 3 times in a row. I will help you as much as possible and im sure that we can sort this out! First, I have some questions: 1. What type of cells did you use? How were they stimulated? 2. What type of controls did you have, how was the results there? 3. What is the difference between well G7 and G8. There seems to be no background in G7, but a lot in G8. Best regards Peter
  23. Dear Staffs, I try to perform B cell ELISPOT and plan to detect antigen (drug)-specific IgG or IgM response. I coated the plate (MultiScreenHTS IP Filter Plate, Millipore) with anti-human IgG mAbs MT91/145 (ref3859-3-250) or anti-human IgM mAbs MT11/12 (3880-3-250), 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). For IgM I have good results but not for IgG I have a big violet background and I am not abble to detect spot. I tried diefferent timing for revelation but it not the problem. I think the concentration of mAb is not good : I used 15µg/mL for coating and 1µg/mL for detection. Do you think it's the problem? I repeated this experiment 3 times. Thanks in advance
  24. ELISpot cell dulitions

    It is smart to begin your project doing some titrations to figure out an optimal cell number for your particular antigen. Maybe responses are much better at 400K cell/well compared to 250K/cells. This is difficult to know prior to actually testing. However, once you have found a cell number where you dont feel limited by a lack of "cell-to-cell" contact, you can stick with that cell number for an entire study. ELISpot and FluoroSpot readers typically have good linearity on counting spots from 0-600 spots. With Mabtech IRIS we see good linearity between 0-2000 spots even beyond. As a result, you are not limited by the read out equipement for capturing both weak and very strong responders.
  25. ELISpot system suitability

    In all situations when you are interested in analyzing cytokine secretion by immune cells. ELISpot/FluoroSpot are reliable and recognized as one of the most sensitive assays among immunological methods.
  26. ELISpot cell dulitions

    Hi all, I understand you often have to plate multiple cell dilutions in the ELISpot to determine the appropriate number of cells that will produce good quality spots. My question is, if all your cell dilutions produce spots, how do you go about deciding which spot counts to use? Do you average them all? Which cell dilution do you "believe"? Thanks!
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