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Christian@mabtech.com last won the day on August 27

Christian@mabtech.com had the most liked content!

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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  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
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