Hello,
I am running a sandwich-type ELISA for which the readout is absorbance at 450nm. The ELISA measures protein binding which I am screening for compounds to inhibit.
The 570nm absorbance value is subtracted from the 450nm value to correct for optical imperfections. My secondary antibody is fused to HRP. Following the final wash step, I add TMB Ultra substrate (Thermo) which is converted by the HRP causing a blue color. The reaction is stopped by the addition of 1M H2SO4, which turns the blue color to yellow.
My recent runs of the ELISA have resulted in absorbance values between 2 and 3. This is in contrast to the initial runs which yielded absorbance values between 0 and 1. Additionally, the hit compound has lost its ability to inhibit signal in recent runs.
In general, for this type of assay, should the maximum absorbance value be 1? Could it be that there is too much protein in the assay to allow for inhibition? What is the optimal absorbance range for these assays and why?
Thank you!
depend on the plate reader, most of them works from 0-3, or 0-4.
the USDA suggests using 0-2, see below.
Optical density range. CVB recommends that the optical density (OD) of
the saturation portion of the ELISA curve not be greater than 2.0 even
though the instrument can measure higher OD values. Since higher ODs
correspond to lower measured signal (transmitted light) in the
spectrophotometer, small variations in the execution of the assay may have a
greater impact at high ODs than at lower ODs.
2.2.2. Reagent blank. The reagent blank should produce ODs of 0.15 or less.
2.2.3. Signal to background (S/B) ratio. The S/B ratio is a key consideration in
determining the optimum reagent working dilutions. An S/B ratio of 10 or
greater when measured near the saturation portion of the ELISA curve is
usually adequate.
No. Look at the mathematical definition of absorbance, and the law of Beer and Lambert. Absorbance and transmitted light are in a logarithmic relationship that allows any value > 0.
In practice, absorbance saturates at high values, because some molecules hide in the "shadows" behind other molecules. There's no light left for them to absorb. In a typical ELISA, you'll see the linear relationship with concentration break down in the range of 1-3 ish -- depending on your molecule, path length and personal tolerance for error.
It's hard to say what's causing your problem. I guess something went wrong with your execution. Sounds like not all the enzyme got removed during the wash steps. You'll need to get your baseline signal back down into the linear range, before you can be sure about the inhibition.
John Schloendorn The concept of molecules "hiding in the shadows" of other molecules is stemming from ray optics - it makes no sense for wave optics and in relation to the question if Beer's law can be applied or not. For details see a recent review on the topic of the Bouguer-Beer-Lambert law: Article The Bouguer-Beer-Lambert Law: Shining Light on the Obscure
Hi,
I am a bit concerned/confused by the statement "The 570nm absorbance value is subtracted from the 450nm value to correct for optical imperfections. "
In general, it is beneficial to be able to see the spectrum, rather than simply rely on a measurement of the loss of light at a single wavelength, as for example scattering can also be registered as a loss of light (absorbance).
What instrument are you using?
Can you measure a spectrum?
If so, how has the spectrum changed between your later and earlier measurements?
Hello Hugh,
Thank you for your answer. I am using a Tecan Spark plate reader. I have only measured absorbance at 450nm and 570nm, not a range.
David Schad
This one? https://lifesciences.tecan.com/multimode-plate-reader
It says it can do spectra - if you can, it is worth recording a spectrum at least at the beginning of a protocol. If there is a lot of scatter, you will see that the peaks are sitting on a large baseline - it is valid to then subtract the absorbance level where the baseline is flat, to give a peak absorbance.
Large scatter could come from, for example, precipitation in the wells.
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