Technical note: Time-resolved immunofluorometric assay for growth hormone in ruminants

P. Løvendahl, J. Adamsen, Regina Teresa Lund, Peter Lind

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    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 muL of sample (plasma or serum) and 200 muL of assay buffer containing 25 ng of mab1.2-Eu3+ conjugate for 4 h at 25degreesC. Plates were then washed six times, incubated for 5 to 10 min with 250 muL 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 (r(2) greater than or equal to 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.
    Original languageEnglish
    JournalJournal of Animal Science
    Volume81
    Issue number5
    Pages (from-to)1294-1299
    ISSN0021-8812
    Publication statusPublished - 2003

    Keywords

    • cervidae
    • bovidae
    • somatotropin
    • radioimmunoassay
    • enzyme-linked immunosorbent assay

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