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  1. 1 point
    Hi E.J. Jung My name is Peter Jahnmatz and I totally agree with Christian, MBC to HBsAg is tricky to find since they are so few in numbers. I have worked with optimisation of a HBsAg MBC assay before. I conducted a study with the similar approach as you were describing. I used the B-cell FluoroSpot assay with peptide-tagged HBsAg-S. Here is a link to that study https://pubmed.ncbi.nlm.nih.gov/31809709/. If you want to, we could have a trouble shooting session and I can show you the reversed approach and the advantages of it compared to coating. Please send me an email to peter@mabtech.com if you are interested. Have a great holiday!
  2. 1 point
    Dear mKim, At Mabtech we sell two enzymatic systems for ELISpot: HRP and ALP ALP in combination with our BCIP/NBT Plus substrate generate spots that are incredibly resilient. No problem having it in the drawer for 2 weeks to validate a new reader. Keep the lid on to protect the wells from getting hair and dust on the membrane. By contrast, HRP spots do degrade over time. The level of degradation depends on how strong and big spots are from the beginning, but in two weeks there can be a some minimal loss of faint spots. After 2-3 months there is clear degradation. Furthermore, HRP spots can be affected by some tap-water, turning yellow post development. As a result, I always prefer ALP over HRP.
  3. 1 point
    Dear Christian, Thank you for your kind support!! Let me contact if I need further supports. Sincerely, Akira
  4. 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.
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