Method of stabilizing enzyme conjugates

In an assay for an analyte having a molecular weight of 100 to 2000, said assay comprising the steps of (a) combining a medium suspected of containing said analyte with a reagent that is a conjugate of an enzyme and a first member of a specific binding pair (sbp) that is an analyte analog and with a second member of a specific binding pair complementary to said first member wherein the enzyme activity of said enzyme is modified upon the binding of said second sbp member to said first sbp member in said conjugate, and (b) determining the enzyme activity of said enzyme by reacting said enzyme with a substrate for said enzyme as an indication of the presence or amount of said analyte, the improvement comprising employing as said reagent said conjugate and an antibody for said enzyme, which antibody is capable of stabilizing said enzyme in said conjugate against thermal inactivation, does not substantially inhibit the activity of said enzyme, and does not substantially inhibit the ability of the first sbp member to bind to said second sbp member, wherein said antibody is employed in a molar amount that is 5-fold or greater than the molar amount of said enzyme conjugate, said amount being sufficient to bind at least 90% of said enzyme conjugate.

A method of determining an analyte having a molecular weight of 100 to 2000 comprising the steps of:

• (a) providing in combination (1) a medium suspected of containing said analyte, (2) a conjugate comprising an enzyme and an analyte analog, (3) an antibody for said enzyme which antibody is capable of stabilizing said enzyme in said conjugate against thermal inactivation, does not substantially inhibit the activity of said enzyme and does not substantially inhibit the ability of said analyte analog to bind to an antibody for said analyte, said antibody for said enzyme being employed in a molar amount that is 5-fold or greater than the molar amount of said conjugate, said amount being sufficient to bind at least 90% of said enzyme conjugate and (4) an antibody for said analyte, which binds to said conjugate and changes the activity of said enzyme, and
• (b) determining the enzyme activity of said enzyme, the enzyme activity being related to the presence or amount of said analyte in said medium.

A composition comprising (1) a conjugate of an enzyme and a member of a specific binding pair (sbp) having a molecular weight of 100 to 2000 and (2) an antibody for said enzyme, which antibody is capable of stabilizing the enzyme in said conjugate against thermal inactivation and does not substantially inhibit the activity of said enzyme and does not substantially inhibit the ability of the sbp member of said conjugate to bind to its complementary sbp member, wherein said antibody is present in said composition in a molar amount that is 5-fold or greater than the molar amount of said conjugate, said amount being sufficient to bind at least 90% of said enzyme conjugate.

A kit comprising in packaged combination (a) composition comprised of (1) a conjugate of an enzyme and a member of a specific binding pair (sbp) having a molecular weight of 100 to 2000 and (2) an antibody for said enzyme, which antibody is capable of stabilizing said enzyme in said conjugate and does not substantially inhibit the activity of said enzyme and does not substantially inhibit the ability of said member to bind to its complementary sbp member, wherein said antibody is present in said composition in a molar amount that is 5-fold or greater than the molar amount of said conjugate, said amount being sufficient to bind at least 90% of said enzyme conjugate, and (b) a substrate for said enzyme.