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  2. ELISpot cell dulitions

    It is smart to begin your project doing some titrations to figure out an optimal cell number for your particular antigen. Maybe responses are much better at 400K cell/well compared to 250K/cells. This is difficult to know prior to actually testing. However, once you have found a cell number where you dont feel limited by a lack of "cell-to-cell" contact, you can stick with that cell number for an entire study. ELISpot and FluoroSpot readers typically have good linearity on counting spots from 0-600 spots. With Mabtech IRIS we see good linearity between 0-2000 spots even beyond. As a result, you are not limited by the read out equipement for capturing both weak and very strong responders.
  3. ELISpot system suitability

    In all situations when you are interested in analyzing cytokine secretion by immune cells. ELISpot/FluoroSpot are reliable and recognized as one of the most sensitive assays among immunological methods.
  4. ELISpot cell dulitions

    Hi all, I understand you often have to plate multiple cell dilutions in the ELISpot to determine the appropriate number of cells that will produce good quality spots. My question is, if all your cell dilutions produce spots, how do you go about deciding which spot counts to use? Do you average them all? Which cell dilution do you "believe"? Thanks!
  5. ELISpot system suitability

    Hi all, what would be some parameters one could use for system suitability of an ELISpot and/or Fluorospot? Thanks!
  6. Antigen-specific B cell ELISPOT

    We are happy to help. Comeback any time Akira! best, Christian
  7. Antigen-specific B cell ELISPOT

    Dear Christian, Thank you for your kind support!! Let me contact if I need further supports. Sincerely, Akira
  8. Antigen-specific B cell ELISPOT

    Dear Akira, Sorry we missed you post! For Human IgM we recommend the following setup: Capture mAbs (MT11/12) at 15ug/ml coating concentration (Code 3880-3-1000) Biotinylated detection mAb (MT22) at 1ug/ml detection (Code 3880-6-1000) And yes, the MT22 detection antibody works fine in Total IgM detection. best, Christian
  9. Mr

    Dear Hari B, Welcome to the forum. Here are my intital reactions/questions: 1. After 5 days stimulation with R848+IL-2, the amount of IgG in the cell cultures is massive. Washing 1x is on the small side. I would recommend doing 3x or 2x and carefully removing all supernatant all the way down to the cell pellet. In this way, free IgG in solution will be reduced which can ofcourse cause high backgorund. 2. The 200,000 cells/well for Total IgG is too high. A better option would be 50,000 cells/well. For your antigen specific wells 200,000 cells/well is ofcourse good. 3. When coating with only PBS, do you include a blocking step in your assay with cell culture medium containing 10% FBS? Blocking of the PVDF membrane is needed. 4. The well names of you uploaded images is not shown to me. My guess is that the really dark ELISpot membrane above is the PBS control, correct?
  10. Mr

    Dear Mabtech, I am optimizing an IgG ELISpot assay for Hep B vaccinated subject. I stimulate my PBMCs from a recently hep B vaccinated subject with R848 and IL2 for 5 days. Wash my cells 1X and resuspend in fresh 10%FCS RPMI before plating 2e5 cells/well. My negative control is PBS coated well (Image B5), positive control is Total IgG (15ug/mL) (image C5) and antigen-specific cells are identified with HBsAg coated (10ug/mL)(image F5) or IgG coated well followed by a biotinylated HBsAg (5ug/mL)(D5). I noticed that the background in my PBS coated well as high. Could you please advise? Thank you.
  11. Antigen-specific B cell ELISPOT

    Dear Jahnmatz, I want to ask one more. To detect total IgM spots, which antibody do you recommend as a coating antibody? I can't find the "Human IgM mAb Guide" in the webpage. Also I appreciate if you can tell me whether "3880-6-250 anti-human IgM mAb MT22, biotinylated" is compatible as a detection antibody in case of detecting total IgM. Thank you for your kind support. Sincerely

    Dear Mr. Cao, Thank you! Please do so, and feel free to post more questions regarding immunoassays. We will try to answer, and if we cannot maybe some other forum guest knows the answer. Take care! Lena

    Dear Lena Beckman Thank you for your reply. I'll try to search on the internet, If i have answer i'll reply here for us refer. Thank you so much. Mr.Cao

    Dear Mr. Cao, Thank you for using our forum! I'm sorry that I haven't responded to you earlier, or that no one else has been able to give you an answer. I've no experience of detecting antibiotics or pesticides. I made a search on the internet and I could find several different suppliers, but I'm no able to recommend any special to you as I'm not familiar with them. I'll ask around in our organization and I hope someone knows, but since our focus is on cytokines, immunoglobulins and apolipoproteins, we are not experts on the detection of antibiotics. I really hope you get the ELISA to work! And please add a reply here on how you solved it, it might help some other researcher. Best regards, Lena
  15. Using Tween20 detergent in Wash Steps of ELISpot

    Dear Del, You have made an important observation! Yes you are right, Mabtech does not recommend Tween and the reason for this is simple: in our hands, with our protocol, it leads to darker ELISpot membranes. There is absolutely no benefit, both for precoated kits and the basic kits where you coat yourself and follow our recommendations on EtOH preactivation! At the same time, other ELISpot practitioners are adamant on the use of Tween as a critical component in making ELISpot results look better. How can our viewpoints be so different? Well, it all comes back to how the plates are set up. We always recommend that you should EtOH treat your plates prior to coating. This increases the binding capacity of the PVDF membrane, and combined with a good amount of capture antibody (1-1.5 ug/well), will always produce better results compared to NOT using EtOH pre-treatment. In a situation where you follow Mabtech's recommendations, Tween provides no benefit and will actually "hurt" your results by making membranes noticeably darker after the plate has been developed. This is true for both the precoated and the freshcoated, but even more noticeable in the latter. However, in cases where you coat your plates yourself and decide not to perform the EtOH pre-treatment, results will actually benefit from the use of Tween. Spots will appear more clear and the "dark-membrane effect" seises to exist, or atleast becomes much less noticeable. It would therefore appear that the use of EtOH fundamentally changes the effect induced by Tween on PVDF membrane. Spots simply look better in my opinion. At the same time, excluding the EtOH activation will make your ELISpot assay suboptimal in terms of sensivitiy. If you buy percoated, plates have been made under optimal conditions so there is no need to use tween.
  16. Hello! When comparing different companies and different protocols for IFNg ELISpots, I've found many using a PBS solution with .5% Tween-20 as an additional wash step. This isn't in your protocol and I've searched for references in regards specifically to your plates (catalog number 3420-2AST-10). Can someone please comment on this? I have always used Tween-20 when using Millipore Nitrocellulose plates and I want to do a comparison with your plates but am afraid of the consequences. Thanks!

    Dear Lena Beckman Thank you for your reply. it's so helpful for me. Thank you so much. About 2 year ago, I was used to ELISA to test some animal disease. But they just teached to me how to used the Elisa kits. But i don't know to setup a elisa kit. I know for the elisa kit we need a: 1. Plate 96 well. 2. We will coat angtigen or antibody dependent on the types of ELISA, incubation and wash. 3. We will add our sample into plate, incubation, wash 4. Add conjugate, incubation, wash, 5. Add substrate, incubation. 6. Add stop solution. And read result assay. I know that plate, conjugate, substrate, stopsolution will by from suppliers. But i don't know where is antigens or antibody in step 2. Can you suggest to me, where i can buy the antigen i need or the ways to have it. This is the firstly time i join to forum, and the first time i setup a elisa kit so there are many thing i don't know! Hope you don't fell annoying. Thank you!

    Dear Mr. Cao, Welcome to our forum! You write that you want to detect pesticides and antibiotics using ELISA, we don’t have any ELISAs or antibodies that can be used for this. Our focus is cytokines, immunoglobulins and apolipoproteins. I can give you some general ELISA information. First I think you should visit our assay principle section on our website, it is very informative and can guide you in how an ELISA assay is working: https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle ELISA is an immunoassay that enables sensitive quantification of analytes in solution e.g. cell supernatants, plasma, serum, CSF, saliva etc. The assay relies on the antibodies that capture and detect the analyte of target, and the antibodies’ affinity, avidity and antigen interactions are essential for the quality of the assay. There are different types of ELISA’s, but we at Mabtech utilized Sandwich ELISA as this is the most sensitive ELISA assay. To set up a Sandwich ELISA you need: · ELISA plates, high protein binding plates, there are several different available, Corning and Nunc are examples of two brands that have this kind of plates. · An antibody pair with specificity against analyte you are interested in. Preferably, the antibody pair should have been validated for ELISA. The antibody pair should contain one capture antibody and one detection antibody. If the detection antibody is biotinylated you will also need an enzyme conjugate like Streptavidin-HRP. Streptavidin binds to the biotin part on the antibody. We have SA-HRP conjugates (link here). Some detection antibodies are directly conjugated with an enzyme e.g. HRP, then the SA-HRP conjugate is not needed. · A substrate that reacts with the enzyme. In the above examples, I have mentioned HRP, and a substrate that reacts with HRP is the TMB ELISA substrate (link here). · TMB substrate needs to be stopped with e.g. H2SO4 (0.2M). · An ELISA reader that can measure the absorbance. · Apart from this you also need several buffers, what buffers that should be used is dependent on the analyte and the antibodies, therefore I cannot recommend any specific. In our ELISAs these are the most common buffers: Coating buffer (PBS pH 7.4). Washing buffer (PBS pH 7.4 with 0.05% Tween), blocking/incubation buffer ( PBS pH 7.4 with 0.1% BSA and 0.05%Tween20) for blocking and dilution of antibodies and samples. I hope this can give you some advice! Good luck! Lena Beckman
  19. Dear Mabtech. I'm a new member. I'm starts setup a new Elisa kit Mabtech can you suggest how to setup a kit elisa to detetion pesticides, antibiotic... I dont' know where is material like antigen, antibodies to coat in plate ? And where are conjugate, and substrate to develope a new elisa kit. Some body can suggest me!
  20. Antigen-specific B cell ELISPOT

    Dear Jahnmatz, Thank you for quick response!! I will follow the recommendation, and please let me contact you again if I need further support. Sincerely,
  21. Antigen-specific B cell ELISPOT

    Hi Akira. I could probably help you with this. 1. The detection antibody for IgG (MT78/145) and IgM (MT22) could be used at 0.5ug/ml (1:1000 dilution). 2. If you noticed that the frequency of antigen-specific B-cells were low, I would recommend using non-sorted PBMC (250-500k cells/well). Since the recovery of purification is not 100%, there is a possibility that you loose important cells in the process. If you want to look at resting MBC, I would recommend pre-stimulation of PBMC for 5 days using R848 and rec.IL-2 before use in the ELISpot. I have attached an article describing this protocol for stimulation (although for FluoroSpot). Dont hesitate to ask if there is something else. Good luck! Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot.pdf
  22. Antigen-specific B cell ELISPOT

    Dear Staffs, This is my first time to perform B cell ELISPOT and plan to detect antigen (drug)-specific IgG or IgM response. My plan is to coat the plate (MultiScreenHTS IP Filter Plate, Millipore) with specific antigen, and use anti-human IgG mAbs MT78/145, biotinylated (3850-6-250) or anti-human IgM mAb MT22, biotinylated (3880-6-250), followed by adding Streptavidin-ALP for ELISpot (3310-10-1000). I am able to detect antigen-specific B cells in PBMC with the similar frequency with flu-specific B cells by FACS, although it is very low frequency. 1. I appreciate if you provide the recommended dilution rate of these Abs to begin with. 2. I wonder if I should use antigen-specific B cell (sorted by FACS) or whole B cells in this ELISPOT. I appreciate if you give the advice for it.. Sincerely,
  23. Stimulation with target cells (co-cultures)

    Hej Esther! Co-cultures of e.g. cancer cells and effector cells is a quite common setup, although we hardly ever perform these experiments in-house. The important thing is that you find a suitable target:effector ratio and to include control wells with e.g. only the target cells to make sure that they don’t produce the cytokines of interest themselves. For protocol inspiration, you can have a look in this paper by Zuber et al 2005: https://www.ncbi.nlm.nih.gov/pubmed/16005014. Although we do not have extensive experience of using adherent cells in ELISpot/FluoroSpot we have not encountered any problems in the few experiments we indeed have made. The cell types we have analyzed include different epithelial cell lines where we have either looked at the production of cytokines (e.g. IL-6 and TNF-alfa) by these cells or have used these cells as APCs. When looking at things secreted by epithelial cells you actually get very nice spots looking very much like spots produced by lymphocytes and there seems to be no interference from possibly remaining cells sticking to the membrane. To my knowledge, there is no special protocol for using adherent cells in ELISpot. We have just used our regular protocols provided in the kits. However, the risk I think one may anticipate when using adherent cells is that they will stick more firmly to the membrane and by not being washed off properly will cause background problems. As said, we have not changed the washing protocol for in our in-house experiments with adherent cells, but logically you could think that some more harsh washing procedures would be required. We normally wash with only PBS in an automatic ELISA washer but one could consider using PBS with 1 mM EDTA and leaving it for some time in the well (most adherent cells would detach under these conditions). I know that some groups also include Tween in their washing buffer but we have never seen any beneficial effect of this but rather that you may sometimes see increased background. Hope you at least got some ideas! Don’t hesitate to come back if you have further questions. Kind regards,Jens
  24. Hej, I want to use the IRIS to check for cytokine production by T cells in response to their target cancer cells. The cancer cells are transduced with the target antigen and our cells are highly responsive to them. Are there protocols available for how to set up co-cultures for the IRIS and is it possible to use adherent cells? Best, Esther
  25. Background Problem with Mouse IFN-gamma ELISPOT assay

    Dear Silvia Pérez! Welcome to the forum. I have looked at your images and made some comments about it in the attached PPT. Please have a look! Overal I think this could be a case of a particular stimuli causing darkening of the membrane. Some peptides induce this effect. I think you could reduce the problem quite a alot by turning off the substrate reaction earlier. Most people tend to overdevelop their plates. Your plate is still wet as can be seen in the photos. Dry it completely and you will see that the membrane does "lighten". You will probably still be able to make out the spots from the dark membrane background. Feedback.pptx
  26. Dear all, I think I'm having the same problem as Christian. I'm doing ELISPOT for detecting IFN-g secreting cells. The conditions are as follow: I use the ELISPOT product code 3321-4APT-2. In each well I add: 80 ul RPMI (+ Gln, 10% FBS, Pen Strep, Sodium Piruvate and 2-mercaptoethanol) 100 ul PPD antigen (diluted in PBS and PBS 0,01%FBS) 50000 cells I incubate the cells ON at 37ºC and 5% CO2. I follow the isntructions regarding washes and incubation times. After adding the substrate, I see wells with a dark grey shadows on the bottom. I don't know how to get rid of them and what is the element is causing this issue. I would appreciate some advice from you. Sincerely, Silvia Pérez
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