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Absorbance Measurement Using A Reference Wavelength

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#1 Bela



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Posted 15 April 2013 - 04:14 AM

Hello all,

I will be performing ELISAs for HBsAg and anti-HCV for the first time. I have a query regarding measuring the absorbance at two different wavelengths, one being the "measurement wavelength" (450nm) and the other "reference wavelength" (620-690nm). We have a Hidex Chameleon MicroPlate Reader in our lab which has both wavelengths 450 & 620. My problem is that I have never measured an ELISA plate using a reference wavelength before and have no idea about how it's done in Hidex Chameleon. I would really appreciate if anyone from the forum members who's done this before in Chameleon could help me out by providing me the protocol for measuring the absorbance in a step wise manner. I know I'm asking too much but I'm really stuck and feeling intimidated just because I don't know how to do it. I tried searching the forum aout this but did not get any useful links. I'm really hoping to find some help from you guys.

Thanks so much for reading.

#2 bob1


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Posted 15 April 2013 - 12:48 PM

Basically the reference is a normalization value that shouldn't change over the plate. This value is subtracted from your measurement reading to give you a standardised reading.

I haven't used your specific plate reader so I can't give you specifics, but you should be able to do a reading at your measurement wavelength and then another at the reference, then take the values and use a calculator or spreadsheet to do the adjustment.

A basic link can be found here

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