Enzyme inhibition assay - Use of fluorescence microplate reader (Jul/20/2006 )
Hi all,
Has anyone ever used a fluorescence microplate reader? we have one in our faculty but no one use it before... I want to perform neuraminidase inhibition assay, can you help me in this?
Has anyone ever used a fluorescence microplate reader? we have one in our faculty but no one use it before... I want to perform neuraminidase inhibition assay, can you help me in this?
I use a fluorescence microplate reader - an FLx800 from Labtech. Certainly the one I use is a very simple setup- halogen blub at one end, photomultiplier at the other end and filters before each of them. Is this similar to what you're using?
I'll help if I can, although I nothing about neuraminidase.
Ben
Thank you, its just the one I would use. Really we have Flex800 but no one use it before and I'm trying to work with... please what is the detection limits of this instrument? is there any spacial notes I should consider when I perform fluorescence assay?
Areej
Hi there,
I'm not entirely sure on the detection limit - I use immobilised substrate, giving a set quantity of fluorophore, so I've never really had to titrate down to determine the LOD. I assume you're using the KC4 software to run your instrument?
The key thing to get right is the sensitivity. The sensitivity range goes from 25-255, and determines the potential applied across the photomultiplier tube - I don't know exactly what this arbitrary scale refers to, but have discovered it’s not a simple linear correlation! Your best bet is to use the auto-sensitivity function to find the sensitivity that puts your fluorescence response in the middle of the relative range (0 - 90, 000 RFU). When you go back to your settings following a reading, the auto-sensitivity box will tell you the sensitivity used, and you can subsequently set this as your sensitivity (taking auto-sensitivity off) so as to ensure all your readings are comparable.
As a rough guide, at a relatively low sensitivity (43), we were able to easily and reproducibly detect a solution of fluorescein at 2 nM (in 35 μL ~ about 500 microns depth).
The only other thing I can think of off the top of my head is to make sure your excitation and emission filters match up as closely as possible to your fluorophore, and wherever possible, use opaque white plates to maximise the fluorescence response.
Hope that is of some use to you!
Ben
Hi Ben, 
Thank you for the valuable notes. Please I have question about the kinetic mode of this reader, are you familiar with this mode and can I incubate the sample for certain time with shaking, without using the dispenser? 
I've done a little bit with the kinetic operating mode.
Unfortunately we don't use (or even have!) temperature control or injection on our model, so I really can't help you on this one- sorry!
Hi Ben,
Please could you tell me how to use auto-sensitivity? I tried that several times but I got error messages!
Thanks
Sorry for the late reply.
Which error message did you get?
To run autosensitivity, you select 'Automatic Sensitivity Adjustment' in the settings options. Normally we scale everything to high wells, so as to set the upper limit. You then enter the well ID for a well that has the highest fluorescence you'll need to measure, and in the "High Value" box, enter a value towards the top end of the range allowed (0-90,000). I normally use 70,000, as this allows for unexpected fluorescence enhancement, etc.
In the 'Starting Sensitivity' box, you need to enter the sensitivity for the the system to start from. The range is 25-255 and controls the potential applied across the PMT (don't ask me exactly what this relationship is!). Typically I just tell it to start at the bottom (25) and work up. The default setting is 100, so if you're getting an error message, it could mean that the starting sensitivity is too high, and you're overloading the PMT (which the instrument really hates, and starts shouting at you!).
Hope this helps.
Ben