title: Development and validation of a radioimmunoassay for fish insulin-like growth factor I (IGF-I) and the effect of aquaculture related stressors on circulating IGF-I levels.
abstract: This paper describes the development and validation of a commercially available radioimmunoassay (RIA) for the detection of fish insulin-like growth factor-I (IGF-I). The assay was developed using recombinant barramundi IGF-I as antigen and recombinant tuna IGF-I as radiolabelled tracer and standard. Assay sensitivity was 0.15 ng/ml, inter-assay variation was 16% (n = 9) and intra-assay variation was 3% (n = 10). Cross reactivity of less than 0.01% was found with salmon insulin, salmon IGF-II and barramundi IGF-II, less than 0.5% with human IGF-I and less than 1% with human IGF-II. Parallel dose-response inhibition curves were shown for barramundi (Lates calcarifer), coho salmon (Oncorhynchus kisutch), Southern Bluefin tuna (Thunnus maccoyii), tilapia (Oreochromis mossambicus), and seabream (Pagrus auratus) IGF-I. The assay was then used to measure stress related changes in different aquacultured fish species. Salt water acclimated Atlantic salmon smolts (Salmo salar) bathed for 2 h in fresh water showed significantly lower IGF-I concentrations than control smolts two days after the bath (53.1 compared to 32.1 ng/ml), with levels of IGF-I also lower in smolts exhibiting stunted growth (stunts). Capture and confinement of wild tuna in sea-cages resulted in a significant decrease in IGF-I levels (28 ng/ml) when compared to tuna captured and sampled immediately (48 ng/ml), but had recovered to starting levels after 3 weeks (43 ng/ml). Handling and isolation in silver perch (Bidyanus bidyanus) led to a gradual decline in IGF-I over a 12 h period (36-19 ng/ml) but showed signs of recovery by 24 h (24 ng/ml) and had recovered fully 72 h after treatment (40 ng/ml). A similar trial in black bream (Acanthopagrus butcherii) showed comparable results with IGF-I levels gradually decreasing (40-26 ng/ml) over 24 h, results that were mirrored by cortisol concentrations which increased during this time (1-26 ng/ml). In the studies presented here changes in IGF-I levels were not observed for at least 3 h after exposure to the stressor. We suggest this is due to the endocrine nature of IGF-I regulation and the clearance rate of IGF-I in vivo.
authors.affiliation: TGR Biosciences, Hindmarsh, SA 5001, Australia. anthony.dyer@tgr-biosciences.com.au
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Dyer
authors.forename: Zee
authors.initials: Z
authors.lastname: Upton
authors.forename: David
authors.initials: D
authors.lastname: Stone
authors.forename: Philip M
authors.initials: PM
authors.lastname: Thomas
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Soole
authors.forename: Naomi
authors.initials: N
authors.lastname: Higgs
authors.forename: Kirsty
authors.initials: K
authors.lastname: Quinn
authors.forename: John F
authors.initials: JF
authors.lastname: Carragher
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D017756:Aquaculture; D003429:Cross Reactions / Q000276:immunology; D005399:Fishes / Q000097:blood* / Q000378:metabolism; D006801:Humans; D006854:Hydrocortisone / Q000097:blood; D007334:Insulin-Like Growth Factor I / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000276:immunology; D010471:Perches / Q000097:blood / Q000378:metabolism; D011863:Radioimmunoassay / Q000379:methods*; D020317:Salmo salar / Q000097:blood / Q000378:metabolism; D013045:Species Specificity; D013312:Stress, Physiological / Q000503:physiopathology*; D013997:Time Factors; D014413:Tuna / Q000097:blood / Q000378:metabolism
chemical_list: D000906:Antibodies; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D006854:Hydrocortisone