Improved biosensor for measurement of progesterone in bovine milk

A biosensor for on-line measurement of progesterone was designed and calibrated. The objectives were to develop an improved immunoassay sensitive enough to measure progesterone in milk at follicular phase concentrations in real-time, and to design a biosensor for implementation of the detection chemistry. Anti-progesterone antibody was coated on small disks of nitrocellulose membrane, which were inserted in the reaction chamber prior to testing. An antigen capture format was used with 5 minutes for binding and peroxidase-labeled progesterone as the competitor, and 3 minutes for substrate development with tetramethylbenzidine as the chromophore. The sensor was designed using micropumps and valves under computer control, and tested fluid volumes of about 1 ml. Output was read as the rate of change in optical density at 645 nm. An automated sampler was designed to draw a test volume at the start of milking from the long milk tube using a 4-way pinch valve. The system could execute a measurement cycle in about 10 min, and progesterone could be measured at concentrations low enough to distinguish mid-luteal phase cows (2-10 ng/ml) from follicular phase cows (<1 ng/ml). The 95% confidence interval for a 0.5 ng/ml reading was estimated to be 0.2 to 1.5 ng/ml.