Method for selective conjugation of analytes to enzymes without unwanted enzyme-enzyme cross-linking

A process of preparing an analyte-enzyme conjugate wherein said enzyme contains one or more free, surface-accessible carboxyl moieties, comprising the steps of:

• selecting a peroxidase, phosphatase, esterase, or galactosidase enzyme comprising at least one free, surface-accessible carboxyl moiety and optionally comprising an active site comprising a carboxyl moiety;
• selecting a blocking agent comprising ammonia, a primary or secondary amine or salts thereof;
• selecting a coupling reagent, that functions to covalently couple an amine to a carboxyl group, wherein when the enzyme comprises an active site comprising a carboxyl moiety the coupling agent is sufficiently bulky to hinder the coupling reagent from reacting with the active site carboxyl moiety;
• treating the selected enzyme with the selected blocking agent and the selected coupling reagent at a pH at which the coupling agent is functional and a temperature at which the enzyme maintains catalytic activity to produce a blocked enzyme, the blocked enzyme having a sufficient number of free, surface-accessible carboxyl moieties blocked with the blocking agent to at least substantially prevent cross-linking between enzyme molecules;
• selecting an analyte molecule comprising a functional group that can be coupled to an amine group;
• and
• conjugating one or more of the selected analyte molecules to an amine group of the blocked enzyme to form the analyte-enzyme conjugate wherein the blocked analyte-enzyme conjugate retains measurable catalytic activity.