Assay for detecting, measuring and monitoring the activities and concentrations of proteins and methods of use thereof

An assay for detecting, measuring or monitoring the activity or concentration of a protein in a test sample, wherein the protein belongs to a plurality of proteins and the plurality of proteins have similar or overlapping properties towards a plurality of substrates, comprising

• adding the plurality of substrates to a plurality of aliquots of the test sample;
• measuring reaction rates between the protein and each substrate;
• determining the activity or the concentration of the protein in the test sample with using a sensitivity coefficient for each substrate and for each protein, wherein the sensitivity coefficient was determined from a sensitivity coefficient sample by
• obtaining a plurality of inhibited dilutions of the sensitivity coefficient sample, wherein the plurality of inhibited dilutions comprise a plurality of concentrations of the protein which are partially to completely inhibited;
• exposing each inhibited dilution of the plurality of inhibited dilutions to each substrate;
• measuring the reaction rates between each uninhibited protein in each inhibited dilution and each substrate;
• calculating the linear relationships between the reaction rates of each uninhibited protein and each concentration of the sensitivity coefficient sample at infinite inhibitor concentration;
• and
• extracting each sensitivity coefficient of each substrate for each protein from the calculated linear relationships.