Methods to measure immunosuppressive tacrolimus, sirolimus, and cyclosporin a complexes in a blood sample

A method of measuring the amount of immunosuppressive complex components, which are specific for an immunosuppressive drug, that are present in a blood sample from an untreated patient in need of immunosuppressive therapy using the immunosuppressive drug, which method comprises the steps of:

• a) lysing a sample of blood obtained from the patient to obtain blood components;
• b) adding to the lysed sample of blood an excess of the immunosuppressive drug to form drug-specific immune complexes;
• c) contacting the mixture of step (b) with a solid surface to which is linked a first antibody, which is specific for an epitope on a blood component in immunosuppressive complex with the immunosuppressant drug, whereupon the immunosuppressive complex is bound by the first antibody linked to the solid surface;
• d) washing to remove any uncomplexed blood components;
• e) contacting immunosuppressive complex bound by the first antibody linked to the solid surface with a second antibody, which is labeled with a detection molecule and which is specific for another epitope on a blood component, which can be the same blood component bound by the first antibody in step (c) or a different blood component in immunosuppressive complex with the immunosuppressant drug, whereupon the immunosuppressive complex is bound by the second antibody;
• f) washing to remove any unbound second antibody;
• and
• g) measuring the amount of detection molecule on the second antibody, which bound to the immunosuppressive complex that was bound by the first antibody in the mixture of step (e), wherein the amount of detection molecule is proportional to the amount of immunosuppressive complex components present in said blood sample that will bind and form immunosuppressive complexes with the immunosuppressive drug.