METHOD FOR SELECTIVE CONJUGATION OF ANALYTES TO ENZYMES WITHOUT UNWANTED ENZYME-ENZYME CROSS-LINKING

摘要

The present invention provides a simple and efficient method of preparing an analyte-enzyme conjugate where the enzyme contains free, surface-accessible carboxyl moieties without generating undesired, cross-linked enzymes, while preserving the functionality of the enzyme. The method involves treating an enzyme with a blocking agent such that the free carboxyl moieties become non - reactive prior to the conjugation reaction with the desired analyte. The yie ld of the analyte-enzyme conjugate and the purity of the conjugates formed are high since cross-linking of the blocked enzymes is minimized or prevented. T he invention is generally useful in preparing any conjugates of an analyte of interest and an enzyme containing surface accessible carboxyl moieties. The invention is particularly useful in preparing conjugates of any analyte of interest and horseradish peroxidase, alkaline phosphatase or acetylcholine esterase. The conjugates prepared according to the invention are useful in a variety of assays including but not limited to enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and the like. The invention is further directed to analyte-enzyme conjugates prepared by the inventive method and t o kits which contain an analyte-enzyme conjugate prepared by the methods herei n for the detection and/or quantitation of an analyte in a sample.