Is it possible to say which is the minimum and maximum absorbance limit in ELISA tests?
Since linearity of transmittance breaks down at higher concentration because of sample saturation and transmitted light at 2Au is less than 1%, can I assume that absorbences higher than 2Au shouldn’t be taken into consideration?
Which value could I consider as minimum limit?
Hello!
To my knowledge, optimal optical density (OD) values for reliable measurements have a distribution similar to a Gauss distribution. Depending on the manufacturer of the UV vis photospectrometer, the optimal range can vary. In my experience 0.1 to 1.0 is the best range; between 1.0 and 1.5 it gets already critical and I always exclude values above 1.5.
The minimum is given automatically by the blank of your analytes.
In case of doubt, double-check with the manuals of your device and kit's instructions for the ELISA.
I agree with Swati: you would first have to check your instrument specifications. It will give you the correct dynamic range you can use.
For ELISA limits, i guess it will depend on your assay. Maybe you can try using the 4PL equation parameters of the standard: bottom (a) would give you the lower limit and top (d) the upper one. But beware of extrapolation since it would not represent what your spectrophotometer really reads.
Hi
Minimum and maximum AU for your ELISA will depend on your calibration curve. But the curve should take some physic into account to be reliable.
Most chemical compound absorb light at some wavelength. Higher concentration of compound is related to higher absorbtion. Absorbance units are log of transmisson. The higest the Absorbance, the smallest the transmission.
Abs > 3 means Transmission < 0.001.
Based on Lamber-Beer Law, the smallest impact of random errors and internal quenching is between 0.1 and 1 Absorbance units.
Starting from 2, the internal quenching can make a plateau in the curve "concentration vs Absorbance".
Absorbance higher than 2 should not be use to calculate concentration except if the calibration curve is well defined in the plateau domain. If you want to include Absorbance >2, be sure that you use a 4P equation. If you work with Absorbance between 0.1 and 1, you can use a linear regression!
Best
When ever I do ELISA, I consider range between 0.05-2.0. Again it depends on ELISA antibodies, some are good at detecting low amount of protein with less background.
I agree with all the colleages, specialy Silvain Giraud and A Henke.
Absorbance is depending on the Lamber-Beer law, and reactives.
In my experience, washes are very important to get acceptable OD from 0.2 to 1.5. Reading time is also important as well as stop the reaction. The longer the time to read the reaction, the higher the absorbances. If a reaction is not stopped it must
be read within 15-20 minutes.
Sorry, I forgot this. If you are getting very high Absorbances, may help that your samples should be diluted. Try serial 1:2 dilutions.
Hi,
thank you all for your answers... but especially Sylvain – this is exactly what I thought, I just wanted to have the confirmation... thank you.
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