ELISA reader at particular nm length absorbs only one color or more ??
EX : at 505 nm length it absorbs pink if i will put yellow color reaction sample its giving high value than pink color. why ????
The readers for ELISA have a special filter for only 5-6 standard wavelengths for ELISA kits(depends from substrate type), You will check your kit instruction with the reader filters(or the substrate electronic absorbance spectrum). http://en.wikipedia.org/wiki/Visible_spectrum
Just put you colored substrate in a spectrophotometer (or have a look in the databases) for the absorbance spectra. Normally you are trying to measure at the maximum to reach the highest sensitivity of your ELISA photometer. All of this ELISA-photometers have a set of filters which fit to the most common substrates. At a first glance it's sufficient to measure the plates a a single wavelength but to reduce background signals from the plate, turbidity etc. a difference measurement at a second wavelength is required. This second wavelength has to be as far away as possible form the absorbance maximum of your substrate.
e.i. TMB --> absorbance maximum at 492 nm, reference wavelength at 620 nm.
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