optical analysis immunoassay

[Incariol]

Five milliliters of blood were withdrawn from each subject by venipuncture into a free-anticoagulant vacuum tube. The blood was immediately centrifuged at 3000 × g for 5 min, and serum was kept frozen at −80 ◦ C until assayed. BDNF serum levels were measured with sandwich-ELISA, using a commercial kit according to the manufacturer’s instructions (Chemicon, USA). Briefly, microtiter plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in sample diluents and stan- dard curve ranged from 7.8 to 500pg of BNDF. Plates were then washed four times with wash buffer, added monoclonal anti-BNDF rabbit antibody (diluted 1:1000 with sample diluents), and incubated for 3h at room temperature. After washing, a second incubation with anti-rabbit antibody peroxidase conjugated (diluted 1:1000) for 1 h at room temperature was carried out. After addition of streptavidin-enzyme, substrate and stop solution, the amount of BDNF was determined (absorbance set in 450 nm). The standard curve demonstrates a direct relation- ship between optical density (OD) and BDNF concentration. Total protein was measured by Lowry’s method using bovine serum albumin as a standard.

*rubs forehead tiredly*

Ok, so its not like I'm stupid, I know what they are doing here. But why, exactly, would I ever want to use bovine serum albumin as a standard in a human serum immunoassay?

Or is this just a place where I need to

at Cunha et al.?

 

[Incariol]

Anyone?

 

[Blayz]

*sigh*. Really, you go to the trouble of posting this but googling "Lowry's method" is beyond you??

From wikipedia:
"The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic protein residues. The Lowry method is best used with protein concentrations of 0.01-1.0 mg/mL. and is based on the reaction of Cu+, produced by the oxidation of peptide bonds"

In other words, Lowry's method measures protein based on it's chemistry. Since all proteins have the same chemistry, it really doesn't matter which proteins you use.

Furthermore, to build a standard curve you need to know both the concentration and readout of the standard samples. Easy to do with BSA which comes in powder form and for which concentration can be accurately measured.

Finally, it was being used as a measure of total protein.