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  2. HOW TO SETUP ELISA KITS

    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
  3. HOW TO SETUP ELISA KITS

    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
  4. HOW TO SETUP ELISA KITS

    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
  5. 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.
  6. 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!
  7. HOW TO SETUP ELISA KITS

    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!
  8. HOW TO SETUP ELISA KITS

    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
  9. 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!
  10. 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,
  11. 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
  12. 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,
  13. 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
  14. 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
  15. 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
  16. 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
  17. Hydrated too many ELISpot PLUS plates

    Hello David! I have personally kept pre-coated plates like this for 48h and then done the assay without any issues. Longer storage times could be theoretically possible but that is nothing we have evaluated ourselves. Furthermore, please keep in mind: - Some bacteria like to break down antibodies. If such a contamination have made it into one of the wells by help of the cell culture medium, this could end bad for the capture antibodies, drastically affecting the assay. - With cell culture medium in all wells you still have evaporation when stored in 8C. Could be a good idea to wrap the edges of the lid with parafilm in order to minimise this effect.
  18. Hi, I miscalculated the number of plates I needed for an experiment and hydrated/blocked too many plates. These pre-coated ELISpot PLUS plates have been washed with DPBS and incubated with serum-containing media for 1 hour at room temperature. Can these be stored and used in the future, and if so for how long? If they can be stored, please share the steps I should take to store them and any steps I should take before using the plates at a later time. Thanks!
  19. Student

    Hi Oliver, Welcome to our Forum! We have discussed your ELISA problem internally here at Mabtech. Generally, it is hard to analyze Protein A as it binds to several antibodies thereby increasing the risk for high background levels. But as you also can detect positive signals in the negative controls, something else must be wrong. We guess that what has happened in your ELISA is a linkage between the coating and the detection antibody. You use an anti-mouse antibody as coating, do you know the origin of the detection antibody? If it is made in mouse the linkage is obvious, but it is also possible that the polyclonal antibody binds the detection antibody due to cross-linkage. There are commercial available ELISAs for the detection of Protein A, unfortunately, we don’t provide such a kit. Let us know if you have any further comments or thoughts. Good Luck! /Lena
  20. Student

    I recently did a sandwich ELISA that failed. We observed positive detection in our negative controls. I was wondering if someone knew where we messed up the ELISA set up. For the ELISA we had a serial dilution of S. aureus culture supernatent as we were trying to detect Protein A given off by S. aureus. We also had a purified Protein A sample that we serially diluted as a positive control. We coated each well with polyclonal rabbit anti-mouse IgG to begin. We then blocked with Bovine Serum Albumin. We then incubated with either S. aureus or Protein A in all by the negative control wells. We then incubated with our secondary antibody which was a monoclonal anti-protein-A antibody conjugated with biotin. We then incubated with streptavadin and peroxidase followed by color development with TMB. This procedure resulted in positive detection in all wells including then negative controls, which had no antigen present.
  21. Directly labeled antigen detection

    Hi again Josh, There is actually a paper, Luque et al 2018, in which the authors utilize a one-step detection system for FluoroSpot: https://www.ncbi.nlm.nih.gov/pubmed/30075182 However, they don't detect fluorophore-labeled antigen, but fluorophore-labeled HLA mulitmeres (thus looking for anti-HLA IgG in a transplantation setting). The HLA multimeres might have a higher binding capacity and bind more fluorophore molecules than a single antigen would, potentially increasing the signal, but at least it's some data showing the feasibility of a one-step detection system. Kind regards, Jens
  22. Directly labeled antigen detection

    Hi Josh, Welcome to the forum! Sounds like an exciting project! I can't say for sure that it would be impossible, but I think the signal loss might be substantial if you were to utilize direcly fluorophore-conjugated antigens. We have done similar experiments in-house, some of which have been published in Jahnmatz et al 2016: https://www.ncbi.nlm.nih.gov/pubmed/26930550. In this paper, we applied a two-step detection system using peptide-tagged antigens and subsequent anti-tag detection with fluorophore-conjugated mAbs (see attached image). The peptide tags - whose amino acid sequences are published in the paper - were recombinantly expressed with the antigen and we developed the fluorophore-labeled anti-tag mAbs, now QC:ed and readily available. We did not even consider testing directly conjugated antigens for three reasons: 1. There is a risk that the fluorophores hide the relevant epitopes of the antigens, essentially rendering them useless. Perhaps that is the issue you have been seeing in the past? A peptide tag can be positioned specifically, e.g. N-terminally and thus won't hinder antibody binding. 2. It can be hard to get consistent fluorophore conjugations on different batches of antigens. With our setup, we can work with large batches of QC:ed fluorophore-labeled antibodies instead. 3. With a peptide-tagged antigen, there is no need to purify the antigen; you can just use the antigen-supernatant. To my knowledge, there are only a few other papers utilizing similar setups, among them Hadjilaou et al 2015: https://www.ncbi.nlm.nih.gov/pubmed/26320246. The authors of this paper used fluorophore-labeled antigen-specific mAbs, and thus also they used a two-step detection setup. Two reasons for taking the Jahnmatz 2016-approach instead is that (i) tagged antigens circumvent that very need for antigen-specific detection mAbs, and (ii) that anti-tag detection mAbs won't compete for binding the same epitopes that the plasma cell secreted antibodies might recognize. The anti-tag mAbs are not yet officially launched, but send me an email (jens@mabtech.com) if you want to continue the discussion and/or would like to try those anti-tag mAbs. Kind regards, Jens
  23. Hi Mabtech, I will be running a fluorospot assay where we will be looking at detecting secreted antibody from plated plasma cells using two isomers of an antigen. In the past we have seen issues with labeling these antigens and I would like to know if we were to directly label with fluorophores what kind of signal loss would we lose in comparison to performing a two step detection? We have tested the antigens via flow cytometry and know of two fluors that work well with them that have excitation/emission in the range necessary for reading. Thanks for your time. Sincerely, Josh
  24. Plate coating

    Hey Sarah, Yes, I have done this many times without any problems. One thing to consider is that the antibody coating solution inside of the wells will start to evaporate even when kept in a cold room with the lid on. So one can wrap the plate in Parafilm to minimize evaporation. Otherwise all liquid could evaporate and leave the membrane exposed to air. Not good. Also, any contaminant that entered into the plate during the plate coating process would get extra time to "grow". So be sure to etoh activate and add capture antibody in sterile conditions. But that is a given ofcourse.
  25. Plate coating

    Hi Mabtech, Have you tested how long the antibody-coated plates last for? Do you know if they would last up to 2-3 days/longer if kept in at 4oC, rather than just overnight? I need to do experiments that can be delayed (at short notice) up to a few days as it depends whether or not samples can arrive on specific days. Therefore, sometimes I will need to plate the capture antibodies 2-3 days in advance so I'm hoping that this won't affect the FluoroSpot assay? Thank you, Sarah
  26. LOW signal with plasma samples

    Hi Sanda, I think your standard curves look good, and the background levels are low. So the assays seem to have worked. The OD signal increases with development time, on both samples and standard, and I think 60 min is a good developing time for pNPP (30 min is a bit too short, why you shouldn't focus on those results). You have diluted the samples with a dilution factor of 2X and 3X, and 3X is often below the lowest standard point and you shouldn't use these measurements. Since you are analyzing plasma samples, you need to dilute the samples at least 2X in the ELISA diluent, so, therefore, you cannot dilute the samples less. The reason why you need to dilute the samples in ELISA diluent is that the diluent prevents false-positive read-outs which may be caused by interference of heterophilic antibodies found in serum and plasma. My guess is that your samples have these very low levels of cytokines. I'm not an expert on PTSD but several factors might affect the levels, like timepoint for sampling, or disease progress etc? Also very important to use a diluent that prevents false-positive read-outs, I hope this has been done previously as well. Best regards, Lena
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