Methods and devices for the detection and/or quantification of an analyte
in a sample are provided. These are positive detection methods and
devices, in that the more analyte is present in the sample, the stronger
the signal that is provided. Devices of the invention include a
mobilization zone including a mobile or mobilizable detectable analyte
analog, a sample application area, primary and secondary capture areas
each including an immobilized binding partner having a binding affinity
for the analyte being tested for a detectable analyte analog. The
mobilization zone, sample application area, primary and secondary capture
area are in fluid continuous contact with each other. In these devices,
the first immobilized binding partner has an equal or lower apparent
affinity for the analyte than it has for the detectable analyte analog.
Methods of this invention involve introducing a sample (which is
suspected of containing the analyte to be tested for) to a device such as
those described herein, and permitting the sample to migrate from the
application area to and through the first and secondary binding zones. A
detectable tracer conjugate is also permitted to migrate through the
device, usually slightly behind the sample so that any analyte in the
sample contacts the first binding partner before the conjugate. Results
of such methods are read based on the presence and/or intensity of the
detectable signal given by conjugate that binds in the second capture
area.