Technical note: time-resolved immunofluorometric assay for growth hormone in ruminants.
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 microL of sample (plasma or serum) and 200 microL of assay buffer containing 25 ng of mab1.2-Eu3+ conjugate for 4 h at 25 degrees C. Plates were then washed six times, incubated for 5 to 10 min with 250 microL 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 > or = 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.
Løvendahl
P
P
Danish Institute of Agricultural Sciences, Department of Animal Breeding and Genetics, DK 8830 Tjele, Denmark. Peter.Lovendahl@agrsci.dk
Adamsen
J
J
Lund
R
R
Lind
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002417:Cattle / Q000097:blood* / Q000276:immunology; D003429:Cross Reactions; D005063:Europium; D015200:Fluoroimmunoassay / Q000379:methods / Q000662:veterinary*; D013006:Growth Hormone / Q000097:blood*; D006736:Horses; D051379:Mice; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D005063:Europium; D013006:Growth Hormone