Biotin conjugates of analytes containing amino, hydroxyl, or thiol functional groups for use in immunodiagnostic assays

A solid phase assay method for detecting vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof in a sample, comprising:

• a) providing a solid phase support having immobilized thereon a protein or antibody that binds to the vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof in the sample, and a compound having Formula I:
• wherein:
• A is —(CH2)q—, —(CH2)qO(CH2)t—, —(CH2)qNR2(CH2)q—, —(CH2)qS(O)u(CH2)q—, —(CH2)tC(O)NR2(CH2)t—, —(CH2)qNR2C(O)(CH2)t—, —(CH2)qNR2C(O)O(CH2)t—, —(CH2)qNR2C(O)NR3(CH2)t—, or —(CH2)qNR2C(O)S(CH2)q—;
• R1 is vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof;
• R2 and R3 are each independently hydrogen or (C1-C6)alkyl;
• m is an integer number selected from 4, 5 and 6;
• n is an integer number selected from 2 to 36;
• q is an integer number selected from 1 to 12;
• t is an integer number selected from 0 to 12;
• and
• u is an integer number selected from 0, 1 and 2;
• b) contacting the solid phase support with the sample of the vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof, and the compound of Formula I, for a time sufficient to allow binding to the protein or antibody attached to the solid phase support;
• c) contacting the solution and solid phase support obtained in step (b) with streptavidin-horse radish peroxidase (SA-HRP), for a time sufficient to effect conjugation of the SA-HRP to the compound of Formula I, which may be unbound or bound to the protein or antibody attached to the solid phase support;
• d) removing the solution obtained in step (c) containing unbound SA-HRP conjugated to the compound of Formula I;
• and
• e) detecting the presence of bound SA-HRP conjugated to the compound of Formula I,
• wherein the amount of the bound SA-HRP conjugated to the compound of Formula I in step e) is inversely proportional to the amount of the vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof in the test sample.

A kit for determining the concentration of vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof in a sample of human serum by an immune-based competitive protein binding assay, comprising:

• a) a standardized quantity of a compound of Formula I or a standardized solution of a compound of Formula I:
• wherein:
• A is —(CH2)q—, —(CH2)qO(CH2)t—, —(CH2)qNR2(CH2)q—, —(CH2)qS(O)u(CH2)q—, —(CH2)tC(O)NR2(CH2)t—, —(CH2)qNR2C(O)(CH2)t—, —(CH2)qNR2C(O)O(CH2)t—, —(CH2)qNR2C(O)NR3(CH2)t—, or —(CH2)qNR2C(O)S(CH2)q—;
• R1 is vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof;
• R2 and R3 are each independently hydrogen or (C1-C6)alkyl;
• m is an integer number selected from 4, 5 and 6;
• n is an integer number selected from 2 to 36;
• q is an integer number selected from 1 to 12;
• t is an integer number selected from 0 to 12;
• and
• u is an integer number selected from 0, 1 and 2;
• and
• b) a standardized quantity of a protein or antibody that specifically binds to vitamin D, thyroxine, estrogen or testosterone or a metabolite or derivative thereof, and to the compound having Formula I, or a pharmaceutically acceptable salt or hydrate thereof.