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Christian@mabtech.com

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Christian@mabtech.com last won the day on April 22

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About Christian@mabtech.com

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    Mabtech representative

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    Accuratly decribe our official policies and recommendations
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    www.mabtech.com

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    Stockholm, Sweden

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  1. 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.
  2. 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
  3. 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.
  4. 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
  5. 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/
  6. 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.
  7. 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.
  8. Antigen-specific B cell ELISPOT

    We are happy to help. Comeback any time Akira! best, Christian
  9. Antigen-specific B cell ELISPOT

    Dear Akira, Sorry we missed you post! For Human IgM we recommend the following setup: Capture mAbs (MT11/12) at 15ug/ml coating concentration (Code 3880-3-1000) Biotinylated detection mAb (MT22) at 1ug/ml detection (Code 3880-6-1000) And yes, the MT22 detection antibody works fine in Total IgM detection. best, Christian
  10. Mr

    Dear Hari B, Welcome to the forum. Here are my intital reactions/questions: 1. After 5 days stimulation with R848+IL-2, the amount of IgG in the cell cultures is massive. Washing 1x is on the small side. I would recommend doing 3x or 2x and carefully removing all supernatant all the way down to the cell pellet. In this way, free IgG in solution will be reduced which can ofcourse cause high backgorund. 2. The 200,000 cells/well for Total IgG is too high. A better option would be 50,000 cells/well. For your antigen specific wells 200,000 cells/well is ofcourse good. 3. When coating with only PBS, do you include a blocking step in your assay with cell culture medium containing 10% FBS? Blocking of the PVDF membrane is needed. 4. The well names of you uploaded images is not shown to me. My guess is that the really dark ELISpot membrane above is the PBS control, correct?
  11. Using Tween20 detergent in Wash Steps of ELISpot

    Dear Del, You have made an important observation! Yes you are right, Mabtech does not recommend Tween and the reason for this is simple: in our hands, with our protocol, it leads to darker ELISpot membranes. There is absolutely no benefit, both for precoated kits and the basic kits where you coat yourself and follow our recommendations on EtOH preactivation! At the same time, other ELISpot practitioners are adamant on the use of Tween as a critical component in making ELISpot results look better. How can our viewpoints be so different? Well, it all comes back to how the plates are set up. We always recommend that you should EtOH treat your plates prior to coating. This increases the binding capacity of the PVDF membrane, and combined with a good amount of capture antibody (1-1.5 ug/well), will always produce better results compared to NOT using EtOH pre-treatment. In a situation where you follow Mabtech's recommendations, Tween provides no benefit and will actually "hurt" your results by making membranes noticeably darker after the plate has been developed. This is true for both the precoated and the freshcoated, but even more noticeable in the latter. However, in cases where you coat your plates yourself and decide not to perform the EtOH pre-treatment, results will actually benefit from the use of Tween. Spots will appear more clear and the "dark-membrane effect" seises to exist, or atleast becomes much less noticeable. It would therefore appear that the use of EtOH fundamentally changes the effect induced by Tween on PVDF membrane. Spots simply look better in my opinion. At the same time, excluding the EtOH activation will make your ELISpot assay suboptimal in terms of sensivitiy. If you buy percoated, plates have been made under optimal conditions so there is no need to use tween.
  12. Background Problem with Mouse IFN-gamma ELISPOT assay

    Dear Silvia PĂ©rez! Welcome to the forum. I have looked at your images and made some comments about it in the attached PPT. Please have a look! Overal I think this could be a case of a particular stimuli causing darkening of the membrane. Some peptides induce this effect. I think you could reduce the problem quite a alot by turning off the substrate reaction earlier. Most people tend to overdevelop their plates. Your plate is still wet as can be seen in the photos. Dry it completely and you will see that the membrane does "lighten". You will probably still be able to make out the spots from the dark membrane background. Feedback.pptx
  13. Hydrated too many ELISpot PLUS plates

    Hello David! I have personally kept pre-coated plates like this for 48h and then done the assay without any issues. Longer storage times could be theoretically possible but that is nothing we have evaluated ourselves. Furthermore, please keep in mind: - Some bacteria like to break down antibodies. If such a contamination have made it into one of the wells by help of the cell culture medium, this could end bad for the capture antibodies, drastically affecting the assay. - With cell culture medium in all wells you still have evaporation when stored in 8C. Could be a good idea to wrap the edges of the lid with parafilm in order to minimise this effect.
  14. Plate coating

    Hey Sarah, Yes, I have done this many times without any problems. One thing to consider is that the antibody coating solution inside of the wells will start to evaporate even when kept in a cold room with the lid on. So one can wrap the plate in Parafilm to minimize evaporation. Otherwise all liquid could evaporate and leave the membrane exposed to air. Not good. Also, any contaminant that entered into the plate during the plate coating process would get extra time to "grow". So be sure to etoh activate and add capture antibody in sterile conditions. But that is a given ofcourse.
  15. LOW signal with plasma samples

    Dear Sanda, How does you standard curve look like? In normal healthy donors you typically have very low levels of IL-10, IL-6, TNFa, IFNg and IL-1beta in plasma. By contrast, in inflammatory diseseas like sepsis for example you can often get above 1000pg IL-6 and IL-10. /Christian
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