Spectrophotometric method for kinetic absorbance measurements in two-phase enzyme immunoassay and apparatus therefor

摘要

A method is described for kinetic measurement of enzyme activity bound to a solid matrix which improves both the sensitivity and speed of one immunoassay method. The immunoassay typically consists of reaction of the analyte with two specific antibodies, one fixed to the surface of a polymeric bead or wall of a test tube, the other added in solution and labeled by covalent coupling to an enzyme. By reaction between analyte and both antibodies, the enzyme-labeled antibody becomes fixed to the surface in a quantity proportional to the quantity of the analyte. After washing sufficiently to remove unreacted enzyme-labeled antibody, fixed enzyme activity is measured by incubation with a substrate and measurement of the rate of the reaction catalyzed. Fixation of the enzyme causes the reaction products to be localized near the surface. To measure the concentration of reactant or product repeatedly during the reaction, the solution must be mixed before each measurement, which can interfere with the measurement. In the prior art, the reaction is stopped after incubation and the product measured once. The method and apparatus disclosed here provides stirring and measurement away from the surface, and thus permits repeated measurement during the reaction. This kinetic assay can be performed more rapidly and sensitively than assays based on a single measurement.