When developing a competitive ELISA how do you improve sensitivity?
The format is to measure polyclonal antibody: a capture mAB on the plate, captures antigen, and then a labelled mAB is incubated with the polyclonal antibody. the lower the signal, the more specific polyclonal antibody there is.
Does the affinity of the tracer mAb affect the sensitivity - in that the higher its affinity for the antigen, the less likely it is to be competed by the antibodies being measured? So for greater sensitivity a lower affinity tracer is better?
Also with a mAB tracer, it would only be competed by those parts of the polyclonal that are sharing the same epitope, the others would not compete. So would a polyclonal tracer be better?
I would imagine such a set up being used to measure serum antibodies?
Yes, a very high affinity detector antibody would be difficult to compete by the antibodies in your sample. And competition, if possible, is not a measure of antibodies content, but only of the presence of antibodies with the same or similar specificity that the mAb you are using. This is a totally biased experiment.
Does the coating mAb also recognizes the antigen? If so, the polyclonal can also release captured antigen, so the system is a bit complicated. Why dont you capture antigen, incubate with the polyclonal antibody and detect bound antibody with a labelled anti-IgG without crossreactivity to the capturing mAb species?
I agree with Gertrudis. It is better to set up a sandwich ELISA. Competitive format has a very narrow dynamic range. Of course, it all depends on your antigen. Small molecular weight antigens may not be suitable for sandwich format.
Given the intrinsic properties of antibody molecules (affinity & avidity), through careful empirical optimization you have a certain degree of latitude but not as much as you may wish. Your realistic option is to try a number of different antibodies to test which combination gives you the sensitivity you need.
While it is usually true that competitive radioimmunoassay will not have a dynamic range that goes too far beyond 2 orders of magnitude but if the system is sensitive enough to serve your needs, there is always the option of diluting more concentrated samples.
Competitive ELISA does not work for polyclonal antibodies in my opinion. Even using polyclonal antibodies for compete, you may potentially only hinder binding to some epitopes which could be targeted only by antibodies in some samples and not in other samples. Thus you can´t really compare ODs of your samples if that is your aim. Why not use an indirect approach or couple your antigen to a linker? Unless you don´t have a pure antigen solution; then I would go for the sandwich approach as well.
Hi Thanks for all the answers, unfortunately I am left with the task of fixing an already developed assay, which in my opinion is not fit for purpose. I have little leeway to change to a different format as the assay already forms part of a regulatory submission. Of course now it is not working. I agree completely, I would prefer an indirect assay, but I am trying to fix the current competitive assay, this being the first time I have worked on a competitive assay. Which is why I was wondering about the importance of the affinity of the tracer antibody. And I had not considered about the polyclonal antibody releasing the captured antigen, another good point.
Hi Alison,
Could you use a polyclonal antibody as the capture antibody and one of the mabs as detection antibody? The polyclonal capture antibody would fix the antigen on all different epitopes and when adding your sample consisting of polyclonal antibody as well they would compete with the capture antibody and release the antigen. This will result in a decrease of signal which is what you want in a competitive ELISA (it does not matter if you only displace the detection antibody or the antigen itself).
Best wishes,
Christiane
Christiane - unfortunately not as most of the assay 'parts' are from a purchased kit.
I have one further question which I would like to see if I have understood the system correctly.
With this assay the outcome we measure is an IC50. Assuming that the polyclonal antibody we test in the assay each time is constant and unchanging, it competes against a purchased biotinlyated polyclonal antibody, if the batch of biotinylated polyclonal antibody changes, and so the affinity of the biotinylated polyclonal changes, then the IC50 measured will be changed as it has to compete more or compete less? Even if what we are testing is unchanged?
Hi Alison,
Per definition the IC50 is a measure of the effectiveness of a substance in inhibiting a specific biological function, in your case the biotinylated polyclonal antibody. So with the competitive ELISA you will determine the IC50 of a polyclonal antibody towards the biotinylated antibody. In my opinion you cannot transfer the IC50 determined in your ELISA to another substance not tested for in the ELISA. So if the affinity of the batch of polyclonal antibody changes the IC50 will change too.
Best wishes,
Christiane
If you use a labelled polyclonal antibody and its properties change (distribution of affinities and fine specificities within the polyclonal mixture), your result will change for sure. You need to use always the same batch or at least to compare extensively every new batch with a reference one.
If you use monoclonal antibodies in the competition assay, their properties will not change, but competition would be focused on a single epitope, so you would not be measuring the total antibody content in your samples, but only a fraction of them (probably minor). If this fraction changes, your results will drastically change, even though the rest of your polyclonal is not so different.
Its complicated, but perhaps you can standardize it as much as possible, given that you cannot design a better assay.
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