Patent ID: 8546095

Claim:
A method for determining the response on calcineurin activity of a human or animal subject to an immunosuppressive therapeutic agent, the method comprising: (i) obtaining an isolated population of T cells from a human or animal subject in receipt of an immunosuppressive therapeutic agent; (ii) lysing the isolated population of T cells; (iii) dividing the lysed isolated T cell population into at least a first aliquot and a second aliquot; (iv) determining a first level of calcineurin activity in the first aliquot of lysed T cells; (v) contacting the second aliquot of lysed T cells with a composition comprising a T cell receptor agonist and then determining a second level of calcineurin activity in the second aliquot of lysed T cells; and (vi) comparing the first and the second levels of calcineurin activity as determined in steps (iv) and (v) respectively, whereby the ratio of the first and the second levels of calcineurin activity indicates the degree of responsiveness on calcineurin activity by the human or animal subject to the immunosuppressive therapeutic agent; wherein the first and the second levels of calcineurin activity are determined by a fluorimetric assay; wherein the fluorimetric assay comprises the steps of: (a) contacting in a reaction mix the first aliquot of lysed T cells and a fluorescently labeled phosphorylated target peptide substrate capable of being dephosphorylated by calcineurin, wherein the target peptide comprises an amino acid sequence of SEQ ID NO.: 2, under conditions allowing calcineurin to dephosphorylate the target peptide; (b) contacting the reaction mix with a titanium oxide matrix, thereby partitioning phosphorylated target peptide from dephosphorylated target peptide; (c) determining a fluorescence intensity of the fluorescently labeled dephosphorylated target peptide; and (d) correlating the fluorescence intensity to the calcineurin activity, said correlating step comprising: (i) providing a test sample, wherein the test sample comprises a known amount of calcineurin activity, and repeating steps (a)-(c) on said test sample, thereby obtaining a value of a test fluorescence intensity corresponding to the known amount of the calcineurin; and (ii) comparing the fluorescence intensity of the fluorescently labeled dephosphorylated target peptide generated from the first aliquot of lysed T cells with the value of the test fluorescence intensity obtained with the known amount of calcineurin, thereby determining an amount of calcineurin activity in the first aliquot of isolated T cells; and (e) repeating steps (a)-(d) for the second aliquot of lysed T cells wherein the target peptide is capable of being specifically dephosphorylated by the β-isoform of calcineurin and comprises SEQ ID NO.: 2, wherein the S-6 position is phosphorylated.