Haptenylated reagents and immunoassays

Water-soluble avidin derivatives of the formula: (Hapten)a-A (I) wherein Hapten is a water-soluble, non-antigenic moiety or group, preferably having a molecular weight of less than 5000, "A" is avidin, "a" is from 1 to 40 and preferably from 10 to 20, and Hapten is covalently bonded to "A" through an amino or carboxyl group thereof or through an azo linkage with a phenol group of a tyrosine or an imidazole group of a histidine group thereof, are reagent compounds for immunoassays. The preferred Haptens are water-soluble substituted aromatic groups having a quaternary amino group, arsonate group or nitro group substituent, or a ring nitrogen group. Kits containing these compounds and antibodies specifically binding with Hapten can be used in immunoassay methods in which the compound of Formula I is contacted with a labeled antiHapten antibody for a time sufficient to permit Hapten-antibody conjugation. The label bound to the avidin derivative or the label present in the unconjugated antibody is then determined. In the preferred method of this invention, the label is an enzyme. The label is then determined by reacting a substrate with the enzyme which is selected to yield a physically measurable product, and measuring the physically measurable product produced thereby. The method can be used in nucleic acid hybridization procedures and immunoassays.