| Autor: |
Englebienne, Patrick, Hoonacker, Anne Van, Verhas, Michel |
| Zdroj: |
Analyst; October 9, 2001, Vol. 126 Issue: 10 p1645-1651, 7p |
| Abstrakt: |
We propose a high-throughput screening method which involves colloidal gold nanoparticles sensitized with the binding protein. Upon interaction with a specific ligand (a polypeptide or a small organic molecule), the surface plasmon resonance absorbance peak of the colloidal gold reagent shifts toward longer wavelengths due to the change in refractive index at the particle surface caused by changes in mass. The shift is proportional to the dose of ligand involved for a fixed amount of binding protein and occurs according to the kinetics of interaction. We applied this property to the analysis of association and dissociation of ligandbinding protein interactions in a small random access clinical chemistry analyzer. The instrument measures the changes in A600 nm over a period of 20 min for each sample. Due to the high degree of automation, the instrument throughput amounts to 144 samples an hour and can be run during 24 h a day in a walk-away mode. When connected to a computer for data handling, a single instrument can consequently handle over 3000 samples a day. Higher throughput instruments are available which can handle as much as ten times more samples. We validated the technique by comparing the affinity constants (range 103−1012 mol−1) calculated for 30 pairs of ligand-protein interactions at different ligand doses with those obtained from other methods, including the BIAcore (slope 0.84; coefficient of correlation r = 0.82). |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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