Related Experiment Video
Updated: Aug 20, 2026
Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay
Published on: January 11, 2017
Validation of the calcineurin phosphatase assay
Pernille B Koefoed-Nielsen1, Nikolaos Karamperis, Kaj Anker Jørgensen
1Department of Renal Medicine C, Research Laboratory, Aarhus University Hospital, Skejby Sygehus, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark. bundgaardpernille@dadlnet.dk
Background:
The calcineurin inhibitors cyclosporine and tacrolimus are used as primary immunosuppressive drugs in transplant patients. Measuring calcineurin phosphatase (CaN) activity is a proposed pharmacodynamic approach to optimize dosing of these drugs.
Methods:
Whole blood samples were obtained from 10 patients treated with calcineurin inhibitors and 20 healthy volunteers and frozen at -80 degrees C. CaN activity was measured by its ability to dephosphorylate a 19-amino acid peptide previously phosphorylated with [gamma-(32)P]ATP. Radioactivity was quantified by liquid scintillation, and results were converted from cpm to U of CaN. Validation of the assay included enzyme kinetics, linearity, precision (at low and normal CaN activities), analytical recovery, and limit of detection.
Results:
The enzyme followed simple Michaelis-Menten-type kinetics: V(max) was estimated as 240 nmol (32)P x L(-1) x min(-1) and K(m) as 70 micromol/L. The assay was linear within the concentration range examined. Analytical recovery varied from 68% to 72%. The total analytical SD was 0.059 and 0.053 U of CaN for high and low CaN activity, respectively. The within-day SD for high and low activity was 0.032 and 0.039 U of CaN, respectively. The limit of detection was 0.04 U of CaN, which is far below the values measured in patients treated with CaN inhibitors.
Conclusions:
In addition to the pharmacokinetic monitoring applied today, the CaN assay can be used to monitor patients treated with calcineurin inhibitors, hopefully leading to prolonged graft survival.
