Polyelectrolytic internal calibration system of a flow-through assay

A method for detecting an analyte within a test sample comprising:

• contacting conjugated probes with a test sample, the conjugated probes comprising detectable probes conjugated with a specific binding member, the conjugated probes being in fluid communication with a porous membrane and the conjugated probes having a net charge prior to contact of the conjugated probes with the test sample, the porous membrane defining a detection zone that contains a capture reagent immobilized therein, at least a portion of the conjugated probes or complexes thereof binding to the capture reagent to generate a detection signal, the porous membrane defining a calibration zone that contains a first polyelectrolyte substantially non-diffusively immobilized therein, conjugated probes that have passed through the detection zone binding to the first polyelectrolyte to generate a calibration signal, the first polyelectrolyte and the conjugated probes binding via ionic interaction between the two;
• and
• comparing the detection signal to the calibration signal to determine the relative amount of analyte present in the test sample.