Thermally induced phase separation immunoassay

Abstract

An immunoassay in which a thermally induced phase separation is used to effect the separation of specifically bound reactants from free reactants is disclosed. A first reactant is conjugated to a temperature-sensitive polymer to form a polymer/reactant conjugate, and a second reactant is conjugated to a reporter to form a reporter/reactant conjugate. The polymer/reactant, reporter/reactant, and biological fluid samples suspected of containing the analyte are admixed in solution at a temperature other than that at which the polymer will precipitate. Specific binding is allowed to occur, thereby forming a ternary complex. The salt concentration of the adjusted solution is then adjusted to a concentration sufficient to cause the complex to precipitate from the solution, the amount of reporter activity in the precipitated complex or in the solution measured and the presence and/or concentration of the analyte therefrom determined. Alternatively, the first reactant may be conjugated to a monomer and subsequently copolymerized with additional monomers to yield a temperature-sensitive copolymer. Multiple analyses may also be performed on a single sample by choosing a variety of polymers, each polymer having a different specific binding partner conjugated thereto and a different critical solution temperature. By altering the temperature and/or the salt concentration of the solution incrementally, the reporter associated with each of the complexes precipitated with each temperature or concentration increment may be measured, and the presence and/or concentration of each of the analytes determined.