Patent ID: 7964363

Claim:
A method for quantitating vitamin D metabolites directly in blood plasma or serum, without the need for prior purification of the vitamin D metabolites, comprising the following steps: (a) adding of an effective amount of a serine protease with endo- and exoproteolytic activity to a sample containing blood plasma or serum and carrying out a digestion of the vitamin D binding proteins in the blood plasma or serum until they can no longer bind any vitamin D metabolites; (b) diluting of said sample containing serine protease, vitamin D metabolites and the digested plasma or serum proteins using a dilution buffer in which the serine protease is substantially inactive; (c) providing of a vitamin D tracer composition which is coupled to a solid phase; (d) providing of a monoclonal antibody against the relevant vitamin D metabolites; (e) combining of the sample with the vitamin D metabolites, the solid phase with the vitamin D tracer composition and the monoclonal antibody, and conducting a competition binding for a pre-determined period of time between vitamin D metabolites, vitamin D tracer bound to a solid phase and monoclonal antibody in a binding buffer in which the serine protease is substantially inactive, (f) separating of the solid phase with the vitamin D tracer compound and bound monoclonal antibody from the binding buffer, and optionally washing of the solid phase; and (g) determining of the amount of monoclonal antibody on the solid phase, and quantitating the vitamin D metabolites in the blood plasma or serum by correlation with standard samples.