title: A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol.
abstract: A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 mul and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity.
authors.affiliation: Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro, 6, 40126 Bologna, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Roda
authors.forename: A C
authors.initials: AC
authors.lastname: Manetta
authors.forename: F
authors.initials: F
authors.lastname: Piazza
authors.forename: P
authors.initials: P
authors.lastname: Simoni
authors.forename: R
authors.initials: R
authors.lastname: Lelli