How long does an antibody take to interact with its target antigen ? When performing an ELISA, why are we "traditionally" incubating antibodies etc for 1 hour or more?
The interaction is usually very quick. You can see the association in Biacore injecting the antigen/antibody during a few minutes (2-3).
And when you add a sample to a blank well (by mistake) in an ELISA plate, the well develops some color at the end, no matters how quickly you try to remove the wrong sample and wash the well.
There are papers describing than 15 minutes for each incubation is more than enough.
Im sure that we are usually incubating longer than required.
But, there is one good practical reason in my opinion for extended times. If the incubations are too short, the difference between wells due to the delay in application could be significant.
The interaction is usually very quick. You can see the association in Biacore injecting the antigen/antibody during a few minutes (2-3).
And when you add a sample to a blank well (by mistake) in an ELISA plate, the well develops some color at the end, no matters how quickly you try to remove the wrong sample and wash the well.
There are papers describing than 15 minutes for each incubation is more than enough.
Im sure that we are usually incubating longer than required.
But, there is one good practical reason in my opinion for extended times. If the incubations are too short, the difference between wells due to the delay in application could be significant.
The interactions are dependent upon a number of factors, including concentrations of both antigen and antibody, affinity of the antibody (low versus high), and temperature. If other parameters (such as concentrations and affinity) are unknown, a longer incubation is likely to increase the chances of success with respect antigen-antibody interactions.
Depends very much on the quality of antibody and the availability of the epitope. If it important for your experiment do biacore or octet measurements. Bear in mind that the affinity says nothing about the on- and off rates of the antibody.
Thanks for your replies.
@Gertrudis: can you point me towards one of these "15min" articles? Many thanks
The best example is an old paper focused on using very short incubations to make the ELISA a faster technique, but I dont have it any more. We discussed it at the lab just because it was far from tradition.
Sachdev Sidhu is using a competitive ELISA to determine the relative affinities of phage clones. While the pre-incubation with the competitor is long, the incubation of complexes on coated plates to capture free antibodies is 15 min (see the title below). This is only an example, but is a case where having a short incubation is good for the purpose of the assay. We have also used the method.
Phage-displayed Antibody Libraries of Synthetic Heavy Chain Complementarity Determining Regions
You could try to reduce your times as much as desired and see the results.
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