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    Standard reconstitution buffer A5

    Thank you very much Christian!
  3. Guest

    Standard reconstitution buffer A5

    Sorry, I meant batch 8 for IL-4
  4. 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.
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    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!
  6. 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
  7. 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
  8. 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.
  9. 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!
  10. 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.
  11. 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.
  12. 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.
  13. 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?
  14. Dear Lucy! This is fantastic news. Easiest to attach things is to put them into a PPT and then upload it.
  15. 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!
  16. 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
  17. Hello, I have an MT57 antibody (product number 3860-3-250) and would like to label it with a fluorescent dye excited by a 488 nm laser and store it in a -80 freezer. I have two questions: 1. I will likely run a buffer exchange. What do you recommend using for the storage buffer? Must it be stored in PBS? Would 10 mM Hepes buffer with 0.1 mM Calcium Chloride with pH 7.5-8 work? 2. Does MT57 contain enough Lysine groups to conjugate it to Alexa 488 NHS Ester? Thanks!3860-3-250.pdf Joseph Rubin
  18. Hey Lucy! For unimmuniced mice you absolutly have to activate B-cell in vitro using our stimulation cocktail. Otherwise, there will be pretty much no spots in the ELISpot. Good luck!
  19. Thanks for your reply Christian - my Mabtech kit just arrived! This is probably a basic question, but if I want to detect total IgG-secreting cells, would i need to activate B cells in vivo? Or is there already a sufficient baseline level of total IgG-secreting cells in unimmunized mice for ELISPOT detection?
  20. Hi again Lucy, Yes, definitly, this could be the reason. Some antibodies simply dont work well in ELISpot. When we develop new antibody systems we can have 8 different pairs that perform well in ELISA. There are differences, but not massive. However, testing the same 8 pairs in ELISpot and only 1or2 works well. ELISpot requires the absolute best antibodies to work well. That is why our portfolio of Mabtech monoclonals tend to be among the best in the market. Buy our kit and repeat!
  21. Thanks for your reply Christian! I am using a monoclonal anti-mouse IgG1 antibody pair from BD - capture #553445 and detection #553441. We had them in the lab already and I thought in theory it should work - but I guess it's not ideal. Do you think this is why the assay didn't work well?
  22. Hi Lucy, welcome to the forum! No this certainly looks strange. Way too low spot count. Where have you purchased the polyclonal antibodies against mouse IgG1? At only 50K cells/well, our in-house results look like this:
  23. Hi there, Thank you to Mabtech for hosting this very helpful forum! I am interested in using ELISPOT to detect antigen-specific B cells after mouse immunization. As a first step I am trying to detect total IgG from polyclonally stimulated C57BL/6 splenocytes. However, I think I am having sensitivity issues with my assay. I do see spots, but far fewer than expected. Annotated images from our AID reader are attached. I used MSIPS4W10 plates that were activated with 35% ethanol and coated overnight with 10 ug/ml capture anti-mouse IgG1. The splenocytes were activated in vitro with
  24. Hey, When you say "blank" wells It still looks to me like these wells contain spots. So all reagents have been added correctly, otherwise there would be zero spots. One feeling I get is that you use tween in your wash buffer? Right or not? Tween is something we do not recommend. It makes reagents penetrate deeper into the PVDF membrane and this is only beneficial if you do NOT etoh activate the membrane. Since you do perform the etoh activation (which is good, sensitivty of the assay increases) tween gives no benefit. However, it can lead to a purple haze in the membrane. Som
  25. Dear all, Recently my collegues and I are experiencing problems with our IFNg ELISpot assays as some wells do not develop properly (and stay white with some spots) - even tough all steps were performed with the correct volume of reagents etc. (this was checked multiple times by different people). I also found the entry of a similar problem in the forum, but our issue could not be resolved by applying the recommended tips. You find a picture of one of our plates attached (see e.g. well E7). We use MSIP plates and activate the membrane as recommended. For development, 100 µL/we
  26. 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.
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