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I'm doing some ELISA development, and I'd like some justification/best practices for background correction. I'm using a horseradish-peroxidase (HRP) detection system along with a TMB substrate and an acid-based stop solution, which is quite common. When TMB is cleaved by HRP, it produces a blue (maximum absorbance (Amax) of 650 nm) product. The addition of the acid not only stops the reaction for endpoint detection, but it amplifies the signal two- to three-fold, and turns the solution yellow, so that it should be read at its Amax of 450 nm. The acid itself supposedly has an Amax of 540 nm, which is why you should use that wavelength for background correction, subtracting it from the A450. Other sources claim the A540 reading is supposed to correct for "optical imperfections" in the plate.

Using either of these explanations, one would assume that the A540 would be relatively uniform across the plate, independent of the A450 of the same well. However, in my experience, that is not the case at all. My blanks and negative controls are averaging about 0.041 absorbance units (AU) at 540 nm, with an average A450 of about 0.065. However, an A450 of 0.9 gives an A540 of 0.051, 1.4 gives 0.6, 2.0 gives 0.73, and 3.5 gives about 0.14. The absorbance spectrum (Fig. 2) of the substrate appears to be quite minimal at 540 nm, so why am I seeing this dose-dependent increase as the A450 increases?

More practically, how should I do background correction? Currently, I'm subtracting each individual well's A540 from its A450. Should I continue this practice, or should I determine an average plate background at 540 nm using my blanks and negative controls, and subtract that value from each individual A450?

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Using different wavelength for reference give some advantages. You can measure a baseline of each well. All well might not have the same baseline because each well might not be the exactly same as others. In addition, you could make scratches on it, you might touch the bottom, or you get some dirt from your bench. And this can be corrected by A540 in your case. So, variations at a lower range are not much concerned. Sometimes, even if you choose a wavelength where the absorbance seems not to change after reaction, there might be a small change. This is not concerned ether, because both absorbances are concentration dependent unless states in solution do not change (froming complexes or something like that).

But still, you get high background from samples showing 1.4 or more at A450. I do not have experiences for this substrate, but you might want to think several possibility: 1)You might get bubbles on the top of your solution in the wells, 2)You might not get accurate values because OD is too high, 3)The solution might get precipitates 4)This may be a very less possibility, but some secondary reaction occurs.

For 2), 3), and 4). You might want to check linearity by drawing a standard curve, and avoid to use the range deviating from the liner relation.

PS I should've mention that you would check the specification of your plate reader. OD=3 could be way to high to measure it. Because light intensity decreases to one thousandth though 1 cm light path at OD=3. It is very hard to measure such weak lights exactly. Probably many specifications of plate readers say you could measure ODs up to around 2, but some people think it is ideal to measure ODs up to around 1.

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