Purification of bovine IGFBP-3 and the development of an enzyme immunoassay for the protein.
Insulin-like growth factor binding protein-3 (IGFBP-3), the most prominent IGF-binding protein in serum, has been demonstrated to modulate the effects of the IGFs but also to exert IGF-independent actions. Quantification of IGFBP-3 in livestock species, in particular ruminants, is commonly limited to blotting methods in spite of the importance of these species. Here we describe the development of a specific homologous enzyme-linked immunosorbent assay (ELISA) to measure bovine IGFBP-3 in bovine plasma, serum and milk. IGFBP-3 purified from bovine serum was used both as standard and also for tracer synthesis. A specific antiserum was raised in rabbits using a synthetic peptide based on the sequence of bovine IGFBP-3. The measuring range of the assay was between 50 and 1000 ng IGFBP-3 per milliliter of plasma or milk. Mean recovery was 97.3% for plasma and 100.1% for milk. Intra- and interassay coefficients of variation were 6.2% and 9.3%, respectively. For the biological verification of the assay, IGFBP-3 was determined in plasma obtained from 12 dairy cows before and after being injected with a depot-formulated growth hormone (GH) preparation. GH, a well-characterized stimulator of IGFBP-3, led to a 1.3-fold increase of basal IGFBP-3 concentrations during days 3 to 19 after the injection. The availability of an ELISA procedure which permits precise and sufficiently sensitive quantification of bovine IGFBP-3 and which can be used on large sample numbers thereby avoiding the need for radioactive labels, should facilitate further research studies.
Institut für Physiologie, Biochemie und Hygiene der Tiere, Rheinische Friedrich Wilhelms-Universität Bonn, Katzenburgweg 7-9, 53115 Bonn, Germany.
Mark
M
Hennies
Helga
H
Sauerwein
D000818:Animals; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification
D018972:Insulin-Like Growth Factor Binding Protein 3