A rapid and sensitive 384-well microtitre format chemiluminescent enzyme immunoassay for 19-nortestosterone.
We developed a competitive chemiluminescent (CL) enzyme immunoassay for rapid, sensitive analysis of 19-nortestosterone (19-NT) in bovine urine. Anti-19-NT polyclonal antibodies were raised in rabbits using a 19-NT-hemisuccinate derivative conjugated with ovalbumin; the derivative was also conjugated with horseradish peroxidase (HRP) as a label. Antibodies were immobilized on 384-well black polystyrene microtitre plates and HRP-labelled 19-NT activity was measured using an efficient chemiluminescent substrate (SuperSignal ELISA Femto) after 3 min incubation. Emitted light was recorded using a conventional, photomultiplier-tube-based microtitre plate reader or a sensitive back-illuminated, cooled CCD camera. The developed method fulfils all the requirements of precision (intra- and inter-assay CV < 10%) and accuracy (mean recovery 94-112%), with a detection limit of 0.03 ppb (1.1 x 10(-9) mol/L) in a urine matrix. Chemiluminescence enhances detectability of the HRP-labelled tracer (thus lowering the limit of detection with respect to colorimetry) and reduces analysis time. The 384-well microtitre plate cuts the sample/reagent volume (20 microL), a five-fold reduction with respect to the conventional 96-well microtitre plate. The developed method is suitable for high-throughput screening of 19-NT in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays.
Roda
Aldo
A
Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro 6, 40126 Bologna, Italy. roda@alma.unibo.it
Manetta
Anna Chiara
AC
Portanti
Ottavio
O
Mirasoli
Mara
M
Guardigli
Massimo
M
Pasini
Patrizia
P
Lelli
Rossella
R
D045930:Anabolic Agents / Q000032:analysis*; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000918:Antibody Specificity; D002417:Cattle; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D006241:Haptens / Q000737:chemistry; D007124:Immunoenzyme Techniques / Q000379:methods*; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D009277:Nandrolone / Q000032:analysis* / Q000276:immunology; D010047:Ovalbumin / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry; D011817:Rabbits / Q000276:immunology
D045930:Anabolic Agents; D000906:Antibodies; D006241:Haptens; D007202:Indicators and Reagents; D009277:Nandrolone; D010047:Ovalbumin; D010544:Peroxidases