Specific wavelenght to find optical density - Specific Wavelenght to determine optical density in ELISA (Sep/10/2007 )

Hello all

I did ELISA. But several questions are emerging in my mind. How to choose the correct wavelenght of light? Pls someone tell me the theroy behind this.

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The selected wavelength depends on which substrate you use. For example, if you use TMB, and stop with acid, read at 450 nm (which is yellow). If you use ABTS, read at 405.
You should be able to find the recommended wavelength for a number of substrates in any standard immunoassay text. Let me know if you can't access such a book and I'll see what I can send you.

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Thank you!

Yes, I used TMB + acid and determined optical density at 450 nm but did not know why?. Thank you once again! I would like to read the book.

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Optimal wavelength are worked out from scanning spectrograms. You don't neccessarily take the highest peak e.g. if there is an interferant peak in the same waveband (most instruments have a spectral bandwidth of several nm) or if there is a strong peak on the "shoulders" of your compound's peak. So you may select the hightest peak that has no interference in the matrix that you use (phos buffer, serum, urine etc). Alternatively, you may use bichromatic techniques that inolve two wavelengths to eliminat ethe effect of interference.

In ELISA systems, you use a detection agent that has been calibrated in the matrix you are using.

It is rare that you would have to identify the optimal wavelength from your own scanning experiments. Rather, you check the literature or manufacturer if you are using kit reagents.

Hope tha thelps.

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Finally, here's that table I was talking about.
[attachment=3606:SUBSTRAT...ND_STOPS.doc]

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