Method for the determination of analyte concentration in a lateral flow sandwich immunoassay exhibiting high-dose hook effect

A method for determining the concentration of an analyte in a fluid test medium susceptible of exhibiting a high-dose hook effect, which comprises:

• a) providing a strip of a test matrix through which the fluid test medium suspected of containing the analyte can flow and which strip has at least two distinct capture bands in which are immobilized antibodies specific to a first epitope of the analyte and which strip bears labeled antibodies whose labels provide a detectable signal and upstream from the capture bands which are specific to a second epitope of the analyte and which are able to flow through the strip along with the fluid test medium upon its application to the strip;
• b) applying the fluid test medium to the strip and allowing it to flow along the strip and carry the labeled antibodies with it to thereby contact the immobilized antibodies in the capture bands and, when sufficient analyte is present in the fluid test medium to partially block binding of the immobilized antibody with the first epitope of the analyte in the first capture band with which the fluid test medium comes into contact as it flows through the strip to form a sandwich of the immobilized antibody, analyte and labeled antibody in the distinct capture hands through which the fluid test medium carries the analyte, the quantity of which sandwich formation is limited by the partial blocking of the immobilized antibody;
• c) quantitatively detecting the signal from the labeled antibody in each of the distinct capture bands in which the sandwich has formed to obtain a pattern of signals, which pattern is unique to the concentration of analyte in the fluid test medium;
• d) mathematically combining the signals from each of the capture bands into the expression Y=g (Y1, Y2, Y3 . . . ) where Y is the overall assay response, g is a mathematical function describing how Y1, Y2 and Y3 . . . are combined and each of Y1, Y2, Y3 . . . are individual responses from each of the individual capture bands;
• e) providing a plot of Y against X where X is a calibrator value for known concentrations of analyte and preparing a dose response curve by solving the equation Y=h(X) where h=the best fitted dose-response curve which is obtained by curve fitting to obtain the best fitted dose response curve thereby creating a monotonous dose-response curve to factor out the blocking between the antibody and the analyte;
• and
• f) calculating the concentration of analyte in the fluid test medium by mathematically combining the results from the capture bands to obtain an overall assay response, Y=Y1+Y2 . . . and calculating the analyte concentration X by solving the equation Y=h(X).