Heterogeneous immunoassays require a means be provided to separate a labeled binding reagent from an unlabeled binding reagent. Frequently, a surface is provided to which is bound a specific binding ligand or receptor. Various surfaces have been used, such as latex beads, which can be filtered; tubes or wells, usually plastic, which also serve as the container for the assay mixture; magnetic particles which can be separated in a magnetic field gradient; insoluble polymers which are separated by centrifugation or are used as the stationary phase of a chromatograph; bibulous materials such as cellulose or glass paper through which reagents can be filtered or transferred by capillary action.
U.S. Pat. No. 4,659,678 describes a method for carrying out an immunoassay in which an antibody labeled with a hapten forms a complex with the analyte in a liquid medium, The complex binds to a solid support to which is bound antibodies to the hapten. Detection of the bound sample is measured using a second antibody to the analyte labeled with reagents such as radioactive iodine, a fluorescer or an enzyme.
U.S. Pat. No. 4,298,685 describes an assay method using antigens covalently linked to an enzyme, a biotinylated antibody and an avidin coated surface. A competitive immunoassay system is described which is suitable for diagnostic assays.
European Patent Application 0 201 079 describes a sandwich immunoassay having biotin attached to one antibody, a second antibody attached to a detectable label and a binding substance for biotin attached to a solid support.
U.S. Pat. No. 4,228,237 describes a method for determining the presence of a ligand in a liquid medium which utilizes enzyme labeled avidin and a biotin labeled reagent. The resulting enzyme activity is related to the quantity of ligand present in the sample.
United Kingdom Application 2 084 317 describes a competitive immunoassay for an antigen which utilizes a solid surface coated with antibody to a hapten or avidin, an antigen-hapten or antigen-biotin conjugate, a soluble antibody to the antigen and an enzyme labeled antibody to the antibody.
U.S. Pat. No. 4,780,423 describes a heterogeneous assay using controlled pore glass particles. The controlled pore glass particles are used in a fluorescent immunoassay as the support for the specific binding partner capable of binding to a ligand. As used in the invention, the glass particles bind a complex of interest, the detection of which is achieved by use of a fluorescent probe. Measurement of fluorescence is carried out in the presence of the glass particles.
The use of solid particles, such as magnetic particles or glass beads, to serve as the support for an immunologic assay is known. An example of such assays include the use of magnetic particles as the solid support in a fluorometric immunoassay as described in U.S. Pat. No. 4,777,145. The use of avidin-coated glass beads in immunoaffinity chromatography and a method for preparing such avidin-coated beads is described by Babashak J. V. and T. M. Phillips, J. of Chromatography 444:21 (1988).
The present invention provides a means to carry out various heterogeneous immunoassays using the improved method of the invention so as to achieve high capacity, rapid binding and convenient washing of the stationary phase of the heterogeneous immunoassay without centrifugation or conventional filtration.