Effect of methanol on the interaction of monoclonal antibody with free and immobilized atrazine studied using the resonant mirror-based biosensor.
The interaction of monoclonal anti-atrazine antibody D6F3 with free and immobilized atrazine was studied using the resonant mirror-based optical biosensor system IAsys. The binding of antibody to atrazine immobilized on silanized surface through albumin spacer was studied in the presence of methanol. The highest affinity was observed in 10% methanol, the kinetic equilibrium association constant KA was 1.16 x 10(9) mol-1 l compared to 4.3 x 10(8) mol-1 l determined in water. The surface binding capacity was 2-fold lower in the presence of methanol compared to aqueous buffer solution. The kinetic rate constants were significantly improved with low contents of methanol; the fastest association and slowest dissociation occurred at 5 and 10% methanol, respectively. The formation of immunocomplexes was observed even in the presence of 50% methanol. To avoid possible disturbing effects resulting from the immobilization of atrazine, the interaction of free atrazine and antibody was studied using the competitive procedure. The determined values of KA were 9.35, 0.73 and 410 x 10(6) mol-1 l for interactions carried out in 10, 30 and 50% methanol, respectively. For practical determination of atrazine using this antibody, the content of 10% methanol in the assay mixture seems to be the best choice. Thirty percent methanol resulted in the lowest affinity; 50% methanol provides the highest affinity, but much smaller signal is measured. The affinity biosensor system IAsys appeared to be a suitable, rapid and convenient tool for studies of binding interactions in the presence of organic solvents.
Skládal
P
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Department of Biochemistry, Masaryk University, Brno, Czech Republic. skladal@chemi.muni.cz
D000911:Antibodies, Monoclonal; D001280:Atrazine / Q000276:immunology*; D015374:Biosensing Techniques; D006540:Herbicides / Q000276:immunology*; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D006540:Herbicides; D001280:Atrazine; D000432:Methanol