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* Danish Institute of Agricultural Sciences, Department of Animal Breeding and Genetics, DK 8830 Tjele, Denmark and
and
Danish Veterinary Institute, Bülowsvej 27, DK 1790 Copenhagen V, Denmark
2 Correspondence:
P.O. Box 50, (phone: +45-8999-1338; fax: +45-8999-1300; E-mail:
Peter.Lovendahl{at}agrsci.dk).
A noncompetitive, time-resolved immunofluorometric assay (TRIFMA) was developed using a selected pair of monoclonal antibodies (mab) raised against recombinant bovine GH, with the catching mab immobilized on microtiter plate wells and the detection mab labeled with Eu3+ as a tracer, arranged as a sandwich. Plates were coated with mab1.15 (680 ng/well) using a phosphate buffer (pH 4.9), and then blocked with assay buffer containing 1% (wt/vol) BSA. The assay procedure involved incubation of 50 µL of sample (plasma or serum) and 200 µL of assay buffer containing 25 ng of mab1.2-Eu3+ conjugate for 4 h at 25°C. Plates were then washed six times, incubated for 5 to 10 min with 250 µL of enhancement solution, and fluorescence read with a time-resolved fluorometer. The sensitivity of the assay was 0.1 ng/mL, and the working range was 0.2 to 200 ng/mL. Recovery of quantitative amounts of bovine GH added to plasma samples was close to 100%. Cross-reactivity with other bovine pituitary hormones or with GH from nonbovidae or cervidae species was not significant. Intra- and interassay CV during routine operation was 4.4 and 10.7%, respectively (mean = 3.54 ng/mL). Plasma concentrations of bovine GH determined by TRIFMA correlated closely (r2 
0.93) with RIA results, with a conversion ratio of 0.62 when the higher specificity of the monoclonal antibodies was taken into account. The TRIFMA is a reliable alternative to RIA methods because the assay employs no radiolabeled or hazardous chemicals, delivers results rapidly, and has little risk of down periods.
Key Words: Bovidae • Cervidae • Enzyme-Linked Immunosorbent Assay • Radioimmunoassay • Somatotropin
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