Functional quantification of cyclosporine A and FK506 in human whole blood by flow cytometry, using the green fluorescent protein as an interleukin-2 reporter gene

Abstract

The concentration of the immunosuppressive drugs cyclosporine A (CSA) and FK506 in biological fluids is routinely determined by antibody-based assays, which for several reasons do not give accurate information on the actual level of immunosuppression in the patient. To alleviate this problem, we developed a functional reporter gene assay which uses the enhancer fragment of the interleukin-2 promoter region driving the expression of the green fluorescent protein (GFP). This construct was stably transfected in the Jurkat human T lymphoblastoid cell line. Upon stimulation of the cell recipient, the GFP was produced and evaluated by flow cytometry. Immunosuppressants acting via inhibition of interleukin-2 synthesis, such as CSA or FK506, inhibited the production of GFP in a dose-dependent manner. This assay can be performed within a working day with a good reproducibility and was more sensitive than the antibody-based assays, since its detection limit was as low as 10 ng/ml for CSA and 0.5 ng/ml for FK506. We used it for the follow up of drug level present in the blood of transplanted patients, and compared the results with those obtained with the antibody-based assay routinely carried out in our hospital. The conclusions suggest that this assay is a valuable alternative to the presently available assays for the measurement of the immunosuppressive activity found in body fluids.