Emory University

Brian R Roberts

Jennifer L Gooch

Jan Pohl

Allan D Kirk

James Tumlin

One aspect of the present disclosure encompasses methods for determining a protein kinase or phosphatase activity in a biological sample, comprising: contacting in a reaction mix a first test sample and a fluorescently-labeled peptide substrate capable of being modified by a protein phosphatase or a protein kinase, contacting the reaction mix with a TiOmatrix, thereby partitioning fluorescently-labeled phosphorylated peptide from fluorescently-labeled dephosphorylated peptide; and determining the fluorescence of the fluorescently-labeled dephosphorylated peptide, thereby determining a protein kinase or phosphatase activity.

Methods for determining calcineurin activity, and uses in predicting therapeutic outcomes

The methods of the disclosure provide fluorescence-based assays for calcineurin activity, especially in isolated T cells. The methods include the stimulation of the T cells with agents that specifically target the TCR with or without influencing co-stimulatory pathways. One TCR agonist is monoclonal antibodies specific for CD3, which more precisely distinguish the inducible activity of calcineurin than does an alternative method targeting the T cell receptor (CD3) combined with CD28 costimulation. This method more accurately distinguishes between the measured level of calcineurin activity of T cells from immunosuppressed transplant recipients and normal individuals, and thus has improved diagnostic accuracy with respect to the response of an individual to immunosuppressant therapy following an organ transplant.

Growing community of inventors

Sandy Springs, GA, United States of America

Brian R Roberts