Fluorescence polarization immunoassays for multiple analytes using sequential addition of tracers

A methodology is presented which relates in general to fluorescence polarization immunoassays (FPIA) and modifications of current processes wherein detection and quantification of several commonly abused or therapeutic drugs in a single biological fluid sample are determined utilizing manual or current software or related equipment to allow the sequential and simultaneous performance of more than one FPIA assay. The methodology involves combining the reagents either separately or pre-mixed, for multiple assays in a single reagent package, these reagents being used to assay quantitative amounts of each of the assay analytes in a sequential step manner. The assay being performed by mixing the sample with a combination reagent and then initiating a specific reaction for each of the separate analytes by sequentially adding a specific reagent, i.e. tracer for each and reading the results during each separate stage to determine the specific reaction taken place and utilization of manual or software modification monitoring to subtract out the contribution to total signal of each specific reagent added previously to the stage of interest.