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.
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.
Taupin
J L
JL
CNRS UMR 5540, Université de Bordeaux II, Cedex, Bordeaux, France. jean-luc.taupin@umr5540.u-bordeaux2.fr
Merville
P
P
McBride
T
T
Potaux
L
L
Moreau
J F
JF
D000911:Antibodies, Monoclonal / Q000276:immunology; D016572:Cyclosporine / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood*; D007376:Interleukin-2 / Q000235:genetics*; D019169:Jurkat Cells; D016030:Kidney Transplantation; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D008775:Methylprednisolone / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016559:Tacrolimus / Q000097:blood*
D000911:Antibodies, Monoclonal; D005938:Glucocorticoids; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016572:Cyclosporine; D016559:Tacrolimus; D008775:Methylprednisolone