Hi all
I recently used a FITC conjugated antibody in a ELISA as secondary antibody followed by anti-goat-HRP as tertiary antibody. However, I got quite week signal in relative to background (the blank background seems OK).
I did some reading and realize that Fluorescein actually may react with hydrogen peroxide, however my chemistry background is not strong enough to figure out if this is the problem in my case.
Can I ask if anyone have tried FTIC conjugated reagent in ELISA based assays or if any one have the knowledge to explain the reaction between FITC and hydrogen peroxide?
Thanks
Hey,
you cannot use FITC labeled antibodies for ELISA. ELISA stands for Enzyme linked immunosorbent assay, means that an enzyme is producing your signal. for example horseradish peroxidase reacts in the signal developing step with a substrate (TMB, or luminol) and hydrogenperoxide. to form either a colored product, which you can measure by UV vis or it produces light (chemiluminisecence).
But you can use FITC labeled Ab's for fluorescence immunoassays, means: your read out is fluorescence. Depending on the format, do not add a substrate, simply excite your plate/array with a light source at around 495 nm and read the fluorescence intensity at around 530 nm. But of cause, the labeled antibody cannot be your capture antibodies (means, the should not be bound to the surface in the first step, otherwise you will just see the same fluoresence everywhere.
Hi Peter, thanks for your answer. I actually used the FITC conjugated antibody as a secondary antibody followed by anti-goat-HRP as tertiary antibody before I add in ELISA substrate.
Ok, that is of cause a different thing! In theory it should work, sorry I confused your question. (I think you mean secondary antibody, I never heard of something like tertiary antibody, The Antibodies which bind to your antigen are called both primary, and the one that binds the primary is called secondary, although it’s the 3rd antibody you are using in a sandwich format)
The Problem is not that the fluorescein reacts with H2O2, in this last step, everything is "fixed" already. I think your problem is, that the tertiary antibody is.
a) not recognizing the secondary very well (Maybe because of the FITC label)
b) The concentration ranges are not well optimized.
Was there an application of those both antibodies together already published? This will help, to sort out, whether this pair works together. You can also try to immobilize your primary antibody on the well of an MTP and make a dilution series of the secondary antibody, then you will see, whether something is binding or not.
Despite from that, I have three suggestions:
In general, if more antibodies come to an ELISA, the less stable is the whole system I would say. What kind of immunoassay format are you applying? I think you do a sandwich assay? You can try the following:
a) Use an already HRP labeled primary Ab, then you avoid this problem. Direct labeling with HRP is also possible and there are Kits as well.
b) Label your primary Ab with Biotin and use Streptavidin-HRP (there are Kits, so that even not chemists can do that)
(Alternative, but not so recommendable: use an anti-FITC-Ab, labeled with HRP as secondary Ab)
c) You already have a labelled Antibody, if you have a microplate reader, which is ready for fluorescence, you can simply use this label. The sensitivity might be a bit lower, but this is not for sure.
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