Receptor assay based on surface plasmon resonance for the assessment of the complex formation activity of cyclosporin A and its metabolites.
OBJECTIVE
A new automated receptor assay has been used to determine the complex formation activity of cyclosporin A (CsA) and its metabolites in whole blood.


METHODS
CsA in vivo forms a complex with cyclophilin A and calcineurin leading to an inhibition of the calmodulin-dependent phosphatase activity of calcineurin. The equilibrium complex formation gives information about the potential immunosuppressive activity of CsA and its metabolites. To measure the amount of this complex the authors developed an automated receptor assay based on an optical biosensor (Biacore) with surface plasmon resonance (SPR) technology.


RESULTS
In the range of 50-300 nM CsA, the intra-day coefficient of variation (CV) was 7.2%, and the inter-day CV was 10.1%. Measuring range of the assay was 10-500 nM with a detection limit of 5 nM and a processing time of 10 min. Recovery rate for sample pretreatment was 74 +/- 5%. 193 blood specimens from heart transplant recipients were analyzed with 3 different methods. The results determined with the receptor assay were correlated with those obtained by fluorescence polarization immunoassay (FPIA; r = 0.599) and high-performance liquid chromatography (HPLC; r = 0.615).


CONCLUSIONS
The receptor assay determines the complex formation activity of CsA and its metabolites with high sensitivity and precision.
Vollenbroeker
B
B
Medizinische Klinik und Poliklinik D, Münster, Germany. b.vollenbroeker@uni-muenster.de
Fobker
M
M
Specht
B
B
Bartetzko
N
N
Erren
M
M
Spener
F
F
Hohage
H
H
D002851:Chromatography, High Pressure Liquid; D021984:Cyclophilin A / Q000378:metabolism; D016572:Cyclosporine / Q000032:analysis* / Q000097:blood / Q000378:metabolism*; D016903:Drug Monitoring / Q000379:methods; D016231:Fluorescence Polarization Immunoassay; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007166:Immunosuppressive Agents / Q000032:analysis* / Q000097:blood / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D007166:Immunosuppressive Agents; D016572:Cyclosporine; C120817:calcineurin phosphatase; D010744:Phosphoric Monoester Hydrolases; D021984:Cyclophilin A