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  2. 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?
  3. 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
  4. Guest

    HBsAg-specific B cell ELISPOT assay

    Hello, Our lab is running an HBsAg-specific B cell ELISPOT in mouse using the Mouse IgG ELISpot BASIC kit (HRP) (3825-2H), however we often don't see any positivity and can't tell if it is due to the assay or lack of response. What positive control would you recommend for this assay? Thank you!
  5. Can a conjugate antibody bind to a well that is not coated with a capture antibody? An empty well not coated with antibody, but did get blocked, went through the steps of conjugate and substrate and was bright yellow, even though the well was never coated and never had a sample in it. Can anyone help explain?
  6. Guest


    Can a conjugate antibody bind to a well that is not coated with a capture antibody? An empty well not coated with antibody, but did get blocked, went through the steps of conjugate and substrate and was bright yellow, even though the well was never coated and never had a sample in it. Can anyone help explain?
  7. Guest

    Determination of Sample Concentration

    Hi Christian, thanks a lot for coming back to me! Yes, for creating the standard curve, I subtract the 'plate blank'. I was just confused because I've got a recommendation from a colleague to also subtract the 'blank (=medium only)' from the measured samples before calculating. But as you said it makes sense to not do that, just wanted to ask experts:) Thanks again for your help! Best, NNN
  8. 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
  9. Hello all, I am planning to conduct dual colour IL-21 and GzmB ELISpots in the future. In order to accomplish this, the detection antibody for IL-21 or GzmB needs to be conjugated with ALP. However, no such products are available in the market. The detection antibody for either cytokine is only found in the biotinylated form and the enzyme is sold separately as Strep-ALP. I'm wondering if it is possible to conjugate biotinlylated IL-21 detection antibody with Strep-ALP before use in ELISpot? Any help would be greatly appreciated. Much thanks, Philip Samaan
  10. 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
  11. Dear Mabtech support Team, I have a question regarding the determination of the sample preparation. So for normalizing the standard curve, I subtract the Blank from all values; but for my samples I also have Blank wells (control/only medium e.g.) measured. So should I also subtract the Blank from my sample values, before using the value for the equation or not? Thanks a lot for your help! Best, NN
  12. Guest

    Standard reconstitution buffer A5

    Thank you very much Christian!
  13. Guest

    Standard reconstitution buffer A5

    Sorry, I meant batch 8 for IL-4
  14. 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.
  15. Guest

    Standard reconstitution buffer A5

    Hi! We bought a Bovine IFNg ELISA BASIC kit (3119-1H-20 batch 15) and a bovine IL4 ELISA Basic kit (3118-1H-20 batch 😎 The datasheet says the kits include 1ml of Standard reconstitution buffer A5. However we didn't receive this tube in any of the kits. How can we reconstitute the recombinant citoquines? Thank you!
  16. 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
  17. Dear Mabtech Customer Service Team! We're performing your protocol for Human IFN-y ELISpot PLUS kit (HRP) on human PBMC with two pools of peptides. Our peptides are SARS-Cov-2 SNMO pool and SARS-Cov-2 S1 pool, we use CD3 for positive control and CD28 as co-stimulatory antibody. We use media RPMI 1640 with 10% FBS for our cells incubation, don't use Tween in our washing buffer, and add all peptides and antibodies directly before incubation of cells in ELISpot wells. Our problem is that in some wells with peptides we saw strange dark clumps, but in control wells and in wells with
  18. Hey there! We unfortunatly do not have antibodies or ELISA kits for detecting Bachem Exenatide. This is not in our R&D pipeline for development. Sorry.
  19. I'm looking for an ELISA kit that could test/detect Bachem Exenatide. I have tried Abcam, MyBiosource, Phoenix, Antibodies online kits, and they aren't able to detect the Bachem Exenatide. If you have done ELISA for Bachem Exenatide, can you please help me? Thank you!
  20. We recommend doing the pre-incubation with at a cell density of 1million PBMC/ml. The vessel can be a 15ml falcon tube but some people prefer the 5ml falcon tubes because the pellet becomes less "concetrated" with a 5ml tube compared to 15ml. The bottom is more cone-shaped with 15ml tubes. It is smart to loosen the top screw abit so that CO2 can get to the cells easily. If the lid is too tight CO2 cannot get in which is bad for the cells.
  21. Guest

    HBsAg-specific B cell ELISPOT assay

    Hi, what culture conditions does Mabtech use for pre-stimulation (cell density and vessel)? I wasn't able to find this information from your papers. Is it also the same for mouse splenocytes? Thank you.
  22. That is unfortunate. I checked our setting and Guest should be allowed to upload attachments, no restrictions. However, I did my own testing and yes, the upload function dont seem to work when posting as a guest. This worked before with earlier versions of the forum software. I will contact our supplier and ask for an update.
  23. Hi Christian, that's what I'm trying to do, but the uploader is stuck on "Loading..." and I can't seem to attach anything?
  24. Dear Lucy! This is fantastic news. Easiest to attach things is to put them into a PPT and then upload it.
  25. Hi again, just wanted to give an update - it worked, thank you so much! I tried to attach some pictures but the editor doesn't seem to be working? In case it's helpful for anyone, I stimulated C57BL/6 splenocytes with 1 ug/mL R848 and 10 ng/mL IL-2 (as recommended) at 2 million cells/mL for 4 days and plated the cells for 24 hours. I strictly followed the Mabtech protocol and used strep-HRP at 1:500 and developed plates with TMB for 10 minutes. Now for the harder task of detecting antigen-specific IgG B cells!
  26. Hi Joseph, It should in principle be possible to label MT57 with Alexa488 using NHS ester. However, we have not opted for this route in the human IgA sandwhich system used in fluorospot. In our FluoroSpot Flex system for human IgA we use MT57 as the capture antibody and MT20 as the detection antibody conjugated with a 490 Fluorophore. https://www.mabtech.com/products/flex/X-06G-1 Regarding storage in different buffers I do not know. Will check with our production unit next week when we are back from easter holiday. With pretty high likelihood, we will not know if this antibody
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