Absorbance Measurement Using A Reference Wavelength - (Apr/15/2013 )
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.
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