FIELD: measuring.
SUBSTANCE: group of inventions relates to methods for assessing affinity of ligands for a specific receptor target molecule. Disclosed is a multiplex method for quantifying binding of a test compound with a predetermined target molecule and with non-target molecules, involving the following steps: (a) obtaining a mixture of target molecules from a crude extract; (b) incubating said mixture of target molecules in a plurality of mixtures of ligands and test compounds; (c) removing unbound ligands from said plurality of target molecules; (d) isolating ligands associated with said target molecules in said mixture of target molecules; (e) determining the amount of said ligand bound by the target molecule using mass spectrometry and a calibration curve; (f) determining the affinity of the test compound for said target molecules; (g) measuring binding of said test compound with a predetermined target molecule and comparing said binding with binding of said test compound with non-target molecules; (h) calculation Kon and Koff said test compound for said target molecule. Also disclosed are multiplex methods for quantifying binding affinity and a multiplex method for determining Kon and/or Koff.
EFFECT: group of inventions provides a multiplex method for assessing binding affinity of test compounds.
26 cl, 6 dwg, 15 tbl, 8 ex
