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Christian@mabtech.com

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Christian@mabtech.com last won the day on December 10 2020

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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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  1. HBsAg-specific B cell ELISPOT assay

    For antigen specific responses it is beneficial to run a 5-day pre-activation culture. Viability of the culture might go down, but the memory B-cells that you are only interested in should still be fine.
  2. HBsAg-specific B cell ELISPOT assay

    Dear E.J. Jung, Looking at your images I do think you have spots when coating with Hepatitis B antigen. However, the spots are rather faint and not so many. Your reader seems to have problem counting these faint spots. Take for example well A4 in figure 4 above. I can with my eyes see that that well contains in the neighbourhood of 30 spots, but you reader is only counting 12. Can you increase the sensitivity of the spot-counting algorithm? Also, do I understand correctly you are running 400K and 200K cells/well? Some antigen specific B-cells are very low in frequency. After 3 days in activation you do not only incubate B-cells into the ELISpot plates. Plenty of other cells tag along and only around 10% of fresh PBMC is B-cells. When coating antigens to PVDF membrane you risk damaging the epitopes of the antigen. They get smushed up against the membrane structure. By reversing the B-cell ELISpot, utilizing biotinylated antigen during the detection phase and using a coated monoclonal to capture all secreted IgG in the bottom, spots often become much nicer looking and more plentiful. Sometimes you can go from having no spots with coated antigen to clear nice responses with biotinylated antigen. Is this something you can try?
  3. How long stable is the SFC on plate membrane after air-dry

    Dear mKim, At Mabtech we sell two enzymatic systems for ELISpot: HRP and ALP ALP in combination with our BCIP/NBT Plus substrate generate spots that are incredibly resilient. No problem having it in the drawer for 2 weeks to validate a new reader. Keep the lid on to protect the wells from getting hair and dust on the membrane. By contrast, HRP spots do degrade over time. The level of degradation depends on how strong and big spots are from the beginning, but in two weeks there can be a some minimal loss of faint spots. After 2-3 months there is clear degradation. Furthermore, HRP spots can be affected by some tap-water, turning yellow post development. As a result, I always prefer ALP over HRP.
  4. IQ/OQ of Eli Scan Plus reader

    Hi, Misook. Our readers are called IRIS and ASTOR. Not Eli Scan Plus. Contrary to what you have heard, Mabtech do indeed offer a very extensive IQ/OQ upon installation. We also validate performance with a PQ. best, Christian
  5. Fluorospot IL-4 positive control

    Hello Leonie, For human IFNg/IL4 I would recommend using either (AntiCD3+antiCD28) or PHA. For IL-4 secretion it is good to go up 100K-150K cells/well in order to get good response. Combining antiCD3 and antiCD28 will give the response a push. Works also without but spots do become stronger and often more plentiful. For PHA I would recommend using the same cell numer. best, Christian
  6. Scientist

    Hi Wendy, welcome to the forum! When the ELISpot substrate sits in its brown bottle there is a low level of precipitate formed inside of the container over time. This can lead to artefacts if you add the substrate directly to an ELISpot plate. By pushing the substrate through a 0.45um filter, you remove precipitates and you avoid this potential problem. You could in theory also use an 0.22um filter for this process, but it builds up a lot of resistance since the smaller filter quickly gets clogged with precipitate. Its doable, but you have to push like crazy if you try to do 12ml in one go. In order to remove antibody aggregates you have to use 0.22um due to their small size. I believe 0.45 will be too large.
  7. 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.
  8. 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.
  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. 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
  12. 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/
  13. 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.
  14. 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.
  15. Antigen-specific B cell ELISPOT

    We are happy to help. Comeback any time Akira! best, Christian
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