Dear all
I want to set up the technique to assess the neuraminidase antiviral resistance in Influenza viruses, using the MUNANA assay.
I already have a TECAN fluorimeter fluorimeter Infinite 200 Pro. I need to get new filters for this technique.
TECAN offers several filters at the emission wavelength of 4-methylumbellyferone. Could you please tell me which of the available filters are you using?
Thanks
Dear Narcís,
Given that the MUNANA assay is based on a fluorophore with the following wavelengths:
- Excitation 350-365 nm
- Emission 440-460 nm
and looking at the spectra in the attached figure that can be found in the following publication (considering a similar behaviour of the fluorophore in your buffer):
Article Design, synthesis and photochemical properties of the first ...
I would recommend the following filters to achieve maximum of sensitivity without incurring in crosstalk:
- Excitation filter at 340 nm with 35 nm of bandwidth
- Emission filter at 465 nm with 35 nm of bandwidth
I would like to add that personally I have no experience with this assay, so maybe further users may give you more hands-on information.
To choose the filters, please remember that the following equation should always be true in order to avoid crosstalk when reading your assay:
EmissionWL - EmissionBW - ExcitationWL - ExcitationBW >5
Where Emission/Excitation WL are the wavelengths of the filter, and Emission/ExcitationBW are the bandwidths of the filter.
Hope this can help you to get started with your project!
Thanks a lot for your suggestion, Nicola.
Tecan has three filters that could fit for this technique:
460 nm ± 10 nm
465 nm ± 35 nm
448 nm ± 7 nm
I'll strongly consider this option, 465 nm ± 35 nm.
Narcís
Glad it was useful!
Hoping to do a welcome thing, here are a couple of further considerations:
If you have access to a monochromator based fluorimeter, it may be worth to do a scan of the fluorophore in your assay, both in excitation and emission, so to have an example of its behavior in your assay and have better knowledge of the actual excitation and emission spectra.
Moreover, the other two filters that you mentioned can also be considered, but it really depends on the intensity of the signal in your assay:
- If you are unsure about the behavior of your assay and you may struggle detecting a good signal, I would opt for the 465+-35 nm. Using the Gain regulation of your instrument you will be then able to optimize the measurement also for stronger signals, and if you actually end up recording strong signals, it opens the doors for further miniaturization of the assay.
- If instead you normally have very strong signals, you can also opt for the other two, with the knowledge that they will work at their best with intense emissions from your assay.
Dear Nicola. Thanks a lot for your suggestions. I'll check the behavior of the reagent at the monochromator.
Narcís
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