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  2. 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?
  3. Hi, I was doing the last step for colour development when I saw many wells turning purple. Does it affect the development of the spots? Thank you. Rongji
  4. 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
  5. I bought 2 products from mabtech.The products code naming 3430-1H-20 and 3420-1H-20 respectively.I want to clarify the cytokines in the plasmaAnd I wonder that what anticoagulants are recommended because it does not mentioned in the instruction book. If I can not centrifuge blood at once how I should store the samples.At room temperature or they are recommended to store in refrigerator at 4-8 degree Celsius? Thank you.
  6. cutegirl

    how to analyse data

    So I should bring all reagents to room temperature every time before I use,right? I bought elisa plate.(costar #2592) i
  7. 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.
  8. cutegirl

    how to analyse data

    thank you very much.I have another questions.I wonder if I should bring all reagents to room temperature before use for every time. And whether a vortex or blow of pipette can be used to dissolve standard powder.
  9. 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.
  10. Guest

    how to analyse data

    Dear Mabtech support Team, I am a postgraduate student in China.I have two questions.Firstly,I have finished my elisa experiments,I wonder which software are recommended to analyse these data. Secondly,I have bought elisa plates 423501 from your company.There are 5 plates together.I know that they can be stored at room temperature.But if I have used one elisa plates,the remained plates should be stored in room temperature or it should be stored in refrigerator. Waiting for your reply.Thanks a lot.
  11. 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
  12. Dear all, I perform IFNg ELISpot with the mabtech reagents following the mabtech protocol and it works great. I have realized there is a small fraction of donors which have a high background in the negative control which prevents showing any significant differences between the negative control and the peptide stimulated wells. Very interestingly, in those donors there are some other viral peptides (presumably the ones the donors have never been in contact with) that are much lower, or where the spot count drops to 0. In other donors that same peptide stimulates T cells properly, the
  13. Hi Idania, I responded in your own thread:
  14. 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
  15. Guest

    Purple spot background

    Hi Christian, We are experience a problem with purple haze background in some wells. We are using the IFNg coating plates from Mabtech, so no need for EtOH activation of the membrane. We are testing human PBMCs response against peptides. Initially, we were washing with PBS as recommended, but, because now we are doing ELISpot after long term culture of the cells, we were having high background in the negative control. Thus, to try to decrease the background, we added 5 wash with PBS-0.05% Tween after cell stimulation following by 5 wash with PBS and we continue using only PBS for the othe
  16. Guest

    Poorly developed ELISpot membrane

    Hi Christian, We are experience a similar problem with the difference that we are using the IFNg coating plates from Mabtech, so we don't EtOH activate the membrane. We are testing human PBMCs response against peptides. Initially, we were using only PBS for washing as recommended, but, because the long term culture of the cells, we were having high background. Thus, to try to decrease the background, we added 5 wash with PBS-0.05% Tween after cell stimulation following by 5 wash with PBS and we continue using only PBS for the other washes. Most of the plates are developing well without th
  17. 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.
  18. Hi, We developed it in-house. There is a monoclonal streptavidin capture antibody coated on the plate (0.5 µg/mL), a polyclonal anti-streptavidin as primary detection antibody (diluted 1:30,000) and a polyclonal conjugated secondary detection antibody (diluted 1:200). No, I don't have signal from the blank when there is no streptavidin.
  19. 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?
  20. Hi Christian, Thank you for answering. Yes it's designed to detect Streptavidin, but we didn't purchase the ELISA from a commercial supplier. The results are better and a bit more consistent in the 96-well plate. We kept the same parameters on the plate reader when changing for the 384-well plate and use also the optimization reading from the machine. The assay was first developped with TMB substrate before changing for chemiluminescence but I could see the same issue of non reproducible data...
  21. 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?
  22. Hi everyone, I have to optimize an ELISA assay and as it's new for me, I'm not very familiar with how to interprate the data I get from my experiments. So far, I don't have a reproducible assay. Each time I run my assay with same materials, same dilutions, same plate, same substrate, same protocole, I always get different results... Either, I get a lower signal than the previous experiment, or my sigmoidal curve on the graph doesn't reach a plateau while I could see it before. Because I get different shape of curves on my graph, I'm not able to calculate the concentration of my sample c
  23. 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
  24. HI All, I am new to this forum and i have a rather basic question and will appreciate your view on this: My question really is - We have an in-house HCP ELISA method in place. The antibody generated against the HCP is being used as coating antibody and biotin conjugation product of the same antibody is used as biotinylated Ab. I would like to you know from you all experts that would using the essentially same Ab (non-conjugated and conjugated with Biotin) could cause any sort competition for the antigen epitope ?? Thanks all in advance
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