Introduction

FK-506 (Tacrolimus) is a microlide lactone of fugal origin with potent immunosuppressive properties. FK-506 is used for primary or rescue immunosuppressant in patients after solid organ transplantation. Clinically relevant FK-506 side effects are nephrotoxicity, neurotoxicity, diabetes mellitus and hypertension. Most of these side effects are more closely related to FK-506 blood levels than dosage, therefore therapeutic drug monitoring of FK-506 levels is a prerequisite for therapy.

Principle of FK-506 Determination

The enzyme immunoassay for FK-506 is based on the competition between the FK-506 to be assayed and the FK-506-Alkaline Phosphatase conjugate, for binding to rabbit antibody directed against FK-506, coated onto microwells. The sample containing the FK-506, and the FK-506-Alkaline Phosphatase conjugate, when added to the microtiter wells, compete for binding to a limiting number of antibody sites. After incubation, each well is rinsed in order to remove non-bound components. The bound enzymatic activity is then measured by the addition of a chromogenic substrate. The intensity of the color developed is inversely proportional to the concentration of FK-506 in the sample. The concentration is calculated on the basis of a standard curve.

Reagents

• 96-well microtiter plate. Twelve strips of 8 detachable wells, coated with Anti-FK-506 antibody.
• One vial of 0.9 ml Negative Calibrator containing 0 ng/ml of FK-506.
• Three vials of Positive Calibrator (0.75 ml) containing 1, 5 and 25 ng/ml of FK-506.
• One bottle containing 10.5 ml of FK506-Alkaline Phosphatase (ACC-ALP) conjugate.
• One bottle containing 10.5 ml of p-Nitrophenyl Phosphate (pNPP) substrate.
• One bottle containing 15 ml of 10xWash Buffer (PBS-Tween).
• One bottle containing 5.5 ml of Stop Solution, 3N NaOH.

Assay Procedure

• Add 25 μl of standard or sample to the bottom of each well.
• Add 100 μl of ACC-ALP conjugate to each well. Incubate at room temperature for 40 minutes.
• Wash microtiter wells 3 times with wash buffer.
• Add 100 μl of pNPP substrate to each well. Incubate at room temperature for 20 min.
• Add 50 μl of stop solution to each well.
• Read absorbance at 405 nm using ELISA reader.

Cross Reactivity

Cross react with Ascomycin. Do not cross react with Gentamicin, Neomycin, Tylosin, Sulfamethazine, Sulfadimethoxine, Zearalenone and Narasin.

FK-506 Inhibition Curve