Hi All.
I have done a sandwich based fluorescent immunoassay. I used a Costar flat bottom black 96 well plate to coat with anti-mouse IgG and the goal was detecting mouse IgG. The sandwich immunoassay was formed by completed by adding anti-mouse IgG-FITC conjugate. I used a micro plate reader to read my fluorescence signal. Interestingly, the signal from blank wells are stronger than the wells with the sample. Can anyone help me find the problem?
The increased florescence signal can be autofluorescnce from the plastic bottom of the blank well?
Thanks.
Hi Narjes
Could you please let me know about the source of all your antibodies (species they were raised in, clonality) and what exactly did your blank comprise of? Normally black plates with transparent bottom for fluorescence measurements are made so that plastic autofluorescence is minimal (we use Greiner flat bottom plates and do observe some plastic autofluorescence in a plate reader and to a lesser extent in a fluorescence microscope). If the signal to noise of the reaction is good then, it usually stands out pretty well.
Could you also share the acquisition parameters such as integration time, exposure and type of readout (bottom/top) that you used in your experiment?
In some cases where the signal from your experimental well is too low, the blank autofluorescence can appear higher. Worst case I would recommend you take your plate to a widefield microscope to check if the ELISA worked.
Hope this helps
Aparajita
Hi Aparajita. Thanks for your answer. I used the goat produced ones (my two antibodies).
My Costar black 96 wll plate is black at bottom as well. Do you think if it is not suitable to use the same plate as the blank?
In my experience, fluorescence measurements are most reliable when excitation and readout is performed at the well bottom. In my hands top measurements did come with the problem of non-consistent values even for the blanks. The antibody source is not apparently a problem, since goat antibodies do not exhibit non specific binding to surfaces.
Regarding your blank I suppose you have both the bait and the tag antibodies without the mouse IgG right? I would include extra controls such as simply uncoated well in the imaging buffer. Also, what medium are you reading out the fluorescence in?
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