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  1. 1 point
    Dear Christian, Thank you for your kind support!! Let me contact if I need further supports. Sincerely, Akira
  2. 1 point
    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.
  3. 1 point
    Lena@mabtech.com

    Antibody coating

    Hi Emma, Yes, I agree with Christian and it is possible to adjust the protocol a bit (but as Christian writes we have not done comparison studies). I sometimes coat in RT (for 3hrs) and block overnight instead, if that suits my experiment schedule better. If you are tight on time, you could instead go for the ELISAPRO kits, with precoated plates. That will save you both time and decrease variability. Link to ELISAPRO IL6: https://www.mabtech.com/products/human-il-6-elisa-pro-kit_3460-1hp-2 Link to ELISAPRO IFNalpha: https://www.mabtech.com/products/human-ifn-alpha-elisa-pro-kit_3425-1hp-2 Good luck! Lena
  4. 1 point
    Hi Priya, Welcome to our forum and thanks for your interest in our products. I will try to answer your questions. 1) The ELISA plates need to be high-protein binding and there are several suppliers available. What plates that are preferred is up to the user. Strip plates are very convenient if you only use a couple of wells for each experiment, but these plates are generally a bit more expensive. Different plates could give different background readings but the difference is generally very minor. If possible (due to the ELISA reader), we recommend to read a reference wavelength (e.g. 650nm), which corresponds to the background from the plastic, subtract the reference OD values from the samples OD reading. 2) For best results, we recommend to coat the plates overnight at +4 degrees. It is possible to store the plates for some more hours but longer periods are not recommended, since this could affect the antibodies. I cannot give an exact time for how long it is possible to have the plates coated and stored before running the assay, this is dependent also on the specific antibodies. When I do freshly coated plates and do not have the time to do the analysis on them after the overnight incubation, I block the plates and put it back into the fridge. I have had plates in the fridge for about 72 hours (including coating and blocking time), and the assay still works. But please, be aware that this is nothing that we recommend. If you need to store plates you should consider pre-coated plates, they are very stable and can be stored for a very long time. 3) This is dependent upon what substrate you are using. If you are using SA-ALP you need a substrate that interacts with ALP, e.g. pNPP substrate (available in our webshop 3652-P10), this substrate requires around 45 to 60 min of developing time, it is possible to do as you write to test different time-points, again this is dependent on your study. pNPP substrate does not need to be stopped and should be read at 405 nm. If you instead are using SA-HRP you will need to have a substrate that interacts with HRP, e.g. TMB substrate (available in our webshop 3652-F10). The development time for TMB substrate is between 10-15 min and it should be stopped with e.g. sulfuric acid and directly read at 450 nm. 4) Concentration of stop solution is dependent upon stop solution used, we usually use sulfuric acid at a concentration of 0.2M. It seems like you are using our ELISA development kits, this is an adaptable and flexible kit that makes it possible for you to set up the assay in your own way. If you are new to ELISA I think you should consider to start with our ELISAPRO kits, these are complete kits with pre-coated plates and everything included for a straightforward assay. Read more here about our different ELISA formats: https://www.mabtech.com/knowledge-center/product-guide/elisa-kits This is another link where we describe the ELISA assay: https://www.mabtech.com/knowledge-center/assay-principles/elisa-assay-principle on the following pages you can also read about how to determine the sample concentration and other ELISA guidelines. Please let me know how things go and if you need any more assistance! Best regards, Lena Beckman
  5. 1 point
    Dear Seong Yeon, welcome to the forum! Yes, the cell count in positive control wells should in most cases be lower compared to the antigen specific wells. In fact, this is often how we do it at Mabech in our quality control for human IFNg, we run: Unstim= 250,000 cells/well CEF= 250,000 cells/well PPD= 250,000 cells/well PHA= 50,000 cells/well AntiCD3= 50,000 cells/well The positive controls have inherently higher spot counts at lower cell numbers simply due to the stimuli being "polyclonal" in its activation. By contrast, antigen specific wells only induce T-cells that are very rare in frequency, so the cell number needs to be higher. Since you are most interested in the antigen specific wells, the unstimulated control should match the cell count to that of the antigen specific wells, not the positive controls. In general it is smart if you initiate your study by doing a small "test" of like 3 donors. Do a titration of cells with your antigen. Test if it is better to incubate 24h or 48h. Include or exclude anti-CD28. The cell numbers above are for human IFNg. By contrast, for a cytokine like IL-17A, it is recommended to use 100,000 cells well in the positive controls as these T-cells are naturally lower in frequency compared to IFNg secreting T-cells. Yes, resting overnight will most often result in differences compared to only 1h. Some people say that you in this way, by resting overnight, “reset” the T-cells into a "tissue specific state" and that this is beneficial for antigen specific responses. I dont have great experience in doing the resting overnight so I cannot comment much. At Mabtech in our QC controls we do the 1h rest mainly due to getting rid of dead/apoptotic cells that would otherwise affect living cells within the culture during the assay. By removing the dead cells the "environment" is improved and you get better T-cell responses.
  6. 1 point
    Dear Tyler, I was probably to fast in my first response. After coming home I have contemplated the situation some more and realised you could very well be right. Antigen coated ELISA plates could maybe work. With coated antigen you typically dont need so much protein bound to the bottom of the well. An ELISA plate might be capable of binding enough antigen for the spots to look good. By contrast, I am certain a total IgG assay in ELISA plate will look horrible. But coated antigen is different. I will tomorrow talk with other people at mabtech and get their input on this situation and I will come back here in the forum. Hold on!
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