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Everything posted by Christian@mabtech.com

  1. A purple tint to the ELISpot membrane after substrate development is rather common. It does not impact performance of the assay. However, maybe you can upload an image so I can have a look?
  2. Hey There, For ELISA we do not have specific requirements on the anticoagulant. Use what you have available. Many cytokines will degrade over time if stored long term in 4-8C. So for studies of cytokines in plasma it is best to store them in -80C. best, Christian
  3. Temperature can have an impact so it can be good to standardize the temperature used for the assay. For human IFNg we recommend to add reconstitution buffer using a pipette and mix thoroughly. Let it dissolve the standard for 5 minutes. Which Mabtech ELISA have you purchased? 423501 is not one of our product codes.
  4. Most ELISA readers comes with a software that allows you to draw graphs of your standard curve and get values for your unknown samples. We at Mabtech use Molecular Devices and their Softmax Pro software. Other users extract aborbance values from the excel export and use Graphpad Prism. Very nice little software that also helps you in statiscal analysis. Precoated ELISA plates from Mabtech should be stored in room temperture.
  5. Dear Josema, I have only experienced this phenomenon in rare instances when others have shared data with me. But it would seem that some peptides, just like you point out, can magically make unstimulated IFNg spots go down in ELISpot assays. Since we are dealing with a living cell culture, the phenomenon can be elusive to understand since it potentially could involve a "bystander effect". For example: - A particular peptide is contaminated with a TLR ligand that sets off a special subset of monocytes that start secreting IL-10. The response will vary in donor to donor depending
  6. Hi Idania, I responded in your own thread:
  7. Dear Idania, I would say in general that you results look very good overall. There would be no difficulty counting this plate accuratly on IRIS or ASTOR. But I see you are using another reader where a tone difference in membrane-color throws off the spot-counting algorithm. I understand the problem. Well D4-D9 are confluent with spots and the substrate often leave a slight purple tone to them. However, the membrane color difference between well D1 and D2 is striking. I think the use of tween can contribute to the problem you are seeing. I therefore would suggest another protoc
  8. You should check if the antibodies by themselves will generate background. Some antibodies are "sticky" and will have problems in sandwhich assays. No streptaividin and signal should be pretty much zero. If not, you know the source of the problem.
  9. Hi, Is the ELISA based on monoclonal antibodies or polyclonal antibodies? Have you developed these in-house? There are many antibodies that generate background because they are "sticky". You screen for this during development. If you exclude strepatividin from your wells, but add all other reagents, do you still get a signal?
  10. Hey Aline, Do I understand correctly that ELISA is designed to detect "streptavidin"? If you purchased the ELISA from a commercial supplier, maybe they can give you some support? Were the result good with the 96 well plate? If yes, then maybe a reading problem with the 384 plate?
  11. Welcome Sudheer! Using the same antibody clone for both capture and detection is possible in ELISA, but only in situations where your antigen is of the correct "format" and it comes with a caveat!: 1. The antigen must be in the form of homodimer so that each molecule has one epitope avaible for binding to the capture, and the same epitope available for binding to the detection mab. It will then work, and homodimer production is not a rare occurance in nature. However, many analytes are heterodimers and then it is gameover. Furthermore, an analyte can be reported to exist in the form
  12. Having contemplated this over the weekend I realized something: - IFNg is secreted by T-cells but also by NK cells and innate lymphoid cells. - By contrast, IL-2 is more T-cell restricted. I have not heard of NK cells or innate immunity cells being able to secrete IL-2 Could it be that you are obeserving an NK cells derived IFNg response as a result of the AAV? That is my best guess. Would it be possible for you to use IL-2 in your study? Mabtech has a great IL-2 system: https://www.mabtech.com/products/mouse-il-2-elispot-basic-kit-kit-alp_3441-2a
  13. The protocol for isolating your splenocytes look fine. Since you are injecting an AAV vector into your mice, it is possible that the vector persist in the cells for long periods of time post injection? Reading a bit on wikipedia about AAV: "AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state" Thus it is not far fetched that TLR7/8 will be stimulated for long periods of time post injection causing IFNg release in NK cells for example. It would be good if you had control mice in your experimental setup were you inject only PBS (and not A
  14. Hi Sajib, Looking at your PPT presentation it is clear to me that these are correct real spots. They are not artefacts, ie they represent true IFNg cytokine secretion. It either comes from something in cell handling or is a true representation of what is going on in vivo in these mice. The positive control ConA in well A4 induces stronger IFNg responses. What is the immunological background of these mice? Something in their biology that could explain a high level of IFNg release in their spleens? How do you isolate the splenocytes? We know from experience that CTL media
  15. Hi Sajib, please start a new thread in the forum and upload some images inside of a PPT. I can give some feedback then!
  16. Dear Keisuke, The great thing about reverse B-cell ELISpot is that the "sticky" Ig phenomenon is pretty much fixed. The biotinylated antigens do not tend to bind randomly to captured IgG. Please note that the reversed B-cell ELISpot approach requires you to coat with anti-IgG in the bottom of the ELISpot plate. We recommend using Mabtech: - Capture mAbs (MT91/145) https://www.mabtech.com/products/human-igg-elispot-basic-kit-kit-alp_3850-2a If you biotinylate your antigens the results can absolutly be read in ELISpot reader. Kits for biotinylating antigens can be pur
  17. Dear Keisuke, In B-cell ELISpot there is this phenomenon we call sticky Ig. In some donors there are a subset of B-cells secreting Ig that just stick to almost anything. This is most common in IgM and IgA secreting B-cells but it does also happen in IgG. The only way to counteract this is to move over to the reversed B-cell ELISpot protocol instead. This is usually the best approach overall for B-cell ELISpot because you often increase the sensitivity of the assay and you consume less antigen. But it requires you to label your antigens with biotin. On the other hand, sticky Ig phenomon ty
  18. Dear Alok, Mabtech use Millipore plates for all our precoated kits. These plates are absolutely standard for ELISpot the Biosys reader should handle them correctly. You should contact their support for assistance. 1. The Mabtech precoated plates should be compatible with the reader. We use the standard PVDF membrane ELISpot plates from Millipore. 2. You should ofcourse use Mabtech IRIS or Mabtech ASTOR Our readers self calibrate on startup så you never have to "plate calibrate" the reader ever again. They use our patent pending RAWspot technology for counting spots which
  19. Hi there! Strong signal in negative wells is a sign of unspecific binding. The blocking may not be complete so that the conjugate binds to the well or there is crossreactivity between the conjugate and some substance in the blocking solution. Which antibodies/system was used?
  20. Hello Kirsten, In all B-cell ELISpots I strongly recommend running total IgG analysis as a positive control. So you have some wells coated with your antigen, and then in others wells were you have coated with anti-IgG. It is good to go down in cell number for the total IgG control since spot numbers will naturally be much higher compared to any antigen wells. If you get spots in total IgG wells you know that the cells are viable and that your ELISpot has worked. If you still then lack spots in you antigen wells it is either due to no antigen specific cells existing or being to
  21. Hello Philip and welcome to the forum! It sounds to me like you are planning on doing a dual ELISpot with the enzymatic approach. We concluded long time ago that it simply does not work. One substrate will generate stronger responses than the other substrate and weak dual positive cells are simply identified as single secreting cells. Furthermore, the readers are rather horrible to use. Mabtech instead invested our time and resources into FluoroSpot. By coupling detection antibodies to fluorophores instead of enzymes correct analysis can be made. Also, with the launch of our o
  22. Hi NN, At Mabtech when we say blank we refer to the the "plate blank" - that is when only substrate and stop solution is added to the wells. This is subtracted by the software before creating the standard curves. We know that not all customers use this kind of blank, but instead chooses to subtract the control with only buffer (incubation buffer or ELISA diluten). Your question, however, concerns whether we recommend subtracting the background values obtained from samples with only medium, e.g. unstimulated control cells. These we do not recommend to subtract, s
  23. Dear Ivana, These batches were sold some time ago before we introduced our ELISA basic formats. Back then the kits were called ELISA development kit and they did not include the A5 reconsititution buffer. However, we kept the product code the same when we moved to ELISA basic. The standard you got in your kit can be reconsituted in pure PBS.
  24. Dear Tetiana, We know from experience that some patient-samples can sometimes generate strange backgrounds in ELISpot that look like the ones seen in your images. One common theme is that the patients analyzed has an ongoing inflammation in their body from a disease state such as cancer, sepsis or autoimmune disease. I have some speculation as to what could be happening: 1. During widespread inflammation cells in the blood become sticky and can form clumps. After Ficoll separation this is often seen when taking out the PBMC band. The inherent stickiness of the cells leads to more deb
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