Strategies for the reversible immobilization of enzymes by use of biotin-bound anti-enzyme antibodies.
Glucose oxidase (E.C. 1.1.3.4) is reversibly immobilized in a reactor coupled to a flow-injection analysis system using an immunological reaction. The antibody used is irreversibly immobilized on the reactor support by an avidin-biotin linkage. The bond between avidin and biotin is nearly irreversible under normal elution conditions for antibody-antigen reactions. The reactor is packed with a support on which avidin is covalently attached and a biotin-bound second antibody is passed over the reactor packing, which immobilizes this antibody. An immune complex of the enzyme, or first anti-enzyme antibody followed separately by enzyme, is introduced into the flow system, resulting in enzyme immobilization. The reactor produced can be used in the determination of 1 x 10(-11) -1 x 10(-6) mole of glucose with a sample size of 20 mul and a sample throughput of 20-30/hr. These results are comparable to or better than those obtained with glucose oxidase directly immobilized on the same support. The enzyme can be removed by elution with low-pH buffers, and the reactor regenerated by injection of the anti-enzyme antibody and the enzyme.
de Alwis
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Department of Chemistry, University of Kansas, Lawrence, KS 66045, U.S.A.
Wilson
G S
GS