Convection during incubation of microplate solid phase immunoassay: Effects on assay response and variation

In solid phase immunoassays, the incubation period is used to specifically bind free analyte molecules of low concentration to immobilized countermolecules. Many of these assays use elevated temperatures during incubation, based on the assumption that the reaction thus is stimulated by enhancing the association constant. In this paper we demonstrate that elevated incubation temperatures are not likely to have any significant effects on the association reaction. Furthermore we show that in many situations, uncontrolled thermally induced free convection plays an important role in mass transport of analyte toward the solid phase. Convection is a function of assay geometry and the properties of the materials used. Our experiments show that it is a major source of interassay variation which never before has been described in the context of immunoassay incubation. Finally we define the conditions under which forced convection will improve assay response and reduce interassay variation. Applying forced convection during sample incubation of a two-step HBsAg assay, we obtained an increase in response rate per time unit by a factor of similar to 5. Interassay variation was reduced from 5-10% to 1-2%. Assay sensitivity improved by a factor of 2-6 without the slightest modification in assay chemistry.