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  3. 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.
  4. HBsAg-specific B cell ELISPOT assay

    Thank you very much for your valuable comments! As you commented on, I am considering reverse ELISPOT. I'd like to ask for additional opinions. Is 5-day culture better than 3-day culture in the pre-stimulation stage? As the incubation time increases, I am concerned that the cell viability may not be good.
  5. HBsAg-specific B cell ELISPOT assay

    Hi E.J. Jung My name is Peter Jahnmatz and I totally agree with Christian, MBC to HBsAg is tricky to find since they are so few in numbers. I have worked with optimisation of a HBsAg MBC assay before. I conducted a study with the similar approach as you were describing. I used the B-cell FluoroSpot assay with peptide-tagged HBsAg-S. Here is a link to that study https://pubmed.ncbi.nlm.nih.gov/31809709/. If you want to, we could have a trouble shooting session and I can show you the reversed approach and the advantages of it compared to coating. Please send me an email to peter@mabtech.com if you are interested. Have a great holiday!
  6. 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?
  7. Dear staff, I am doing HBsAg-specific B cell ELISPOT assay using your Human IgG ELISPOT Basic (3850-2H), and the plate is Millipore MSIPS4510 (PVDF menbrane)(3654-TP-10). The goal of the experiment is to evaluate the HBsAg-specific B cell response in PBMCs before and after HBV vaccination in humans without HBsAb. I am currently conducting a preliminary experiment, but the HBsAg specific B cell response is not clear (spot is hardly visible), so I would like to discuss the cause of the problem. The preliminary experiment was conducted by randomly drawing the blood of the researchers to properly establish the protocol, including the concentration of antigen, without comparing PBMCs before and after hepatitis B vaccine vaccination. When referring to the HBsAg specific B cell ELISPOT assay protocol performed in another laboratory, pre-stimulation was performed for 5 days, not 3 days, so the stimulation was performed for 5 days with 1 ug/mL R848 and 10 ng/mL IL-2, but no spot was observed (figure1).(one subject's PBMC sample, triplicates, Ag concentration 5, 7, 10ug/mL) I thought that incubation for 5 days might have a negative effect on cell viability, so I performed stimulation for 5 days (figure2) and stimulation for 3 days (figure3) again, but as shown in the figure, several wells were not stained(two subject's PBMC samples, duplicates, 0.4M and 0.2M). Although accurate comparison was difficult, there was no difference between the 3-day culture and 5-day culture, and it was thought that the antigen concentration of 10 ug/mL would be appropriate. As shown in figures 2 and 3, the problem of not being dyed was thought to be an abnormality in the plate (the manufacturing date is unknown, but it was purchased 4 years ago). The same plate was newly ordered, and PBMC were stimulated for 3 days, and the HBsAg was 10ug/mL, and the response cell was 0.2M, again (figure 4). The problem of non-staining was no longer found, but the Ag specific B cell response seemed to be indistinct (spots are almost 0-10 levels), so the experiment was performed again by raising the Ag concentration from 10 to 20 ug/mL (figure 5). But again, the spot doesn't seem to be much different. What is the reason the spot is barely visible? Looking at the PC, it seems that there is no problem with PBMC or stimulation. All of the PBMCs used in the preliminary experiment belong to those who have been vaccinated against hepatitis B vaccine and have antibodies. I am asking for opinions as I am not sure what the problem is. Thank you. E.J. Jung
  8. Thank you very much for prompt reply! Your information helped me a lot. HRP is more familiar for experiment and I use it generally. I will use ALP kit next time.
  9. 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.
  10. I have measured the IFNg using ELISpot after overnight air-dry. However, the device will be replaced and I have to prove the equivalence of the results between the old and a new device. If it is long stability of the spots on the plate membrane experimented 1 or 2 weeks ago, I would like to keep the membrane at dark(RT) and I measure it again using the new device. Could you give the information of the stability of the signal on dried membrane or advice for bioequivalence test by the change of device? Thank you in advance!
  11. IQ/OQ of Eli Scan Plus reader

    I was confused the model. Thank you for prompt reply
  12. 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
  13. IQ/OQ of Eli Scan Plus reader

    I work at clinical laboratory in Korea and my company bought your Eli Scan Plus reader. I am going to use it for a project(clinical trial) in several weeks later. One customer (pharmaceutical company) related to the project asked its IQ and OQ. However I heard that you do not provide IQ/OQ for the reader. How could I prepare for documentation instead of IQ/OQ? or If it is not necessary for the reader, could you explain/ provide the evidence?
  14. 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
  15. Hello, I would like to perform the IFNg/IL-4 Fluorospot on PBMCs stimulated with a vaccine antigen in order to see if the vaccine induces predominantly a Th1 or Th2 response. Do you have a recommenation for a positive control for IL-4 secretion? E.g. PBMCs of a patient with a specific helminth infection, malaria,...? Thanks! Leonie
  16. Indeed I plate the cells first - I add 100 µL cells and then 10 µL of the stimulus. I will try adding the stimulus in 50 µL medium first. Thank you very much for the quick support!
  17. Blank areas in IFNg ELISpot with human PBMCs

    Oh, I see! It looks like the cells have been "pushed" to the side of the well. That might happen if you add the cell suspension first and the stimulus after. In what order are you adding reagents? The best way is to first add medium including (or not including) the stimuli and the cell suspension last. Use for example 50% of the final medium volume for the stimuli (or just medium for negative control) and 50% of the final volume for the cell suspension.
  18. Hello Jens, Sorry, maybe I should have been clearer, but I am not concerned by completely empty wells, but by the "pattern" that is seen in some wells. So some areas in the certain wells that show no spots or very few and the cells seem to be pushed outside. I wash the plate with a multipipette and I am rather careful, so I don't think it is caused by too aggressive washing. Thank you, Lena
  19. Blank areas in IFNg ELISpot with human PBMCs

    Hej Lena! It looks like you are experiencing the exact same issue as has been discussed in this thread: Briefly, probably some reagent have not reached the bottom of the well. We have seen these blank wells a couple of times, and we have yet to experience a blank well that cannot be "rescued" by repeating the detection steps again. Thus, the problem is not a lack of coated capture antibodies, but a one failed detection step. Please repeat the detection steps in the blank wells as follows: 1. Wash the blank wells 5x with 200ul PBS. 2. Add detection antibody at 1ug/ml in PBS containing 0.5% FCS and incubate for 2h. 3. Wash the blank wells 5x with 200ul PBS. 4. Add SA-ALP in PBS containing 0.5% FCS and incubate for 1h. 5. Wash the blank wells 5x with 200ul PBS. 6. Add substrate and incubate for 10min. I would be surprised if you didn't get results looking like this: So what to do to avoid blank wells in future experiments? Well, if you are too careful with emptying the pipette, there is a risk for a bubble formation in the bottom of the well, that blocks the following reagent to reach the bottom. Solution: •Look carefully at the plate for odd looking wells after addition of liquid. •Remove bubbles by tapping the plate against the bench. •Do not touch inner wall of the well with the pipette tip. Hope you manage to resolve the issue! Please come back to us if you don't (or, yeah, please do if you resolve it as well, would be nice for us to know :))
  20. Scientist

    Thank-you Christian
  21. Hello! I have been conducting IFNg ELISpot assays with human PBMCs for a while now, but recently I am observing blank areas in my wells (either no spots at all or much less than in the rest of the well) - it can be seen in most of the wells of the attached picture. I tried changing the ethanol-treatment of the membrane, putting the cells through a cell strainer before plating etc., but nothing seems to help. I don't really know what else to do, especially as the spot distribution was great 2 months ago using the same protocol. Would could cause this and how can I solve this issue? Thank you very much in advance, Lena
  22. 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.
  23. Scientist

    In the human IFN-gamma plus kit, it suggests 0.22 um filter for the primary antibody solution, and 0.45 um filter for substrate. I am guessing the primary filtration would be to remove aggregates? What is the reason for filtering substrate? would 0.22 work for the substrate as well? or 0.45 work for the primary antibody filtration step. thank-you for the information!
  24. 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.
  25. 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
  26. I am Using the AID ELISpot reading machine
  27. 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
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