Establishment of time-resolved fluoroimmunoassays for detection of growth hormone and insulin-like growth factor I in rainbow trout plasma

Comp Biochem Physiol A Mol Integr Physiol. 2020 Oct:248:110751. doi: 10.1016/j.cbpa.2020.110751. Epub 2020 Jul 4.

Abstract

The GH/IGF-I axis influences many aspects of salmonid life history and is involved in a variety of physiological processes that are related to somatic growth (e.g., reproduction, smoltification, and the response to fasting and stress). As such, fisheries studies utilize GH/IGF-I axis components as indicators of growth and metabolic status. This study established time-resolved fluoroimmunoassays (TR-FIAs) for rainbow trout plasma GH and IGF-I using commercially available reagents. For the GH TR-FIA, the ED80 and ED20 were 0.6 and 28.1 ng/mL, the minimum detection limit was 0.2 ng/mL, and the intra- and inter-assay coefficients of variation (%CV) were 4.1% and 13.4%, respectively. Ethanol remaining from acid-ethanol cryoprecipitation (AEC) of plasma samples to remove IGF binding proteins reduced binding and increased variability in the IGF-I TR-FIA. Drying down and reconstituting extracted samples restored binding and reduced variability. The extraction efficiency of IGF-I standards through AEC, drying down, and reconstitution did not vary over the working range of the assay. For the IGF-I TR-FIA, the ED80 and ED20 were 0.2 and 6.5 ng/mL, the minimum detection limit was 0.03 ng/mL, and the intra- and inter-assay %CV were 3.0% and 6.5%, respectively. Biological validation was provided by GH injection and fasting studies in rainbow trout. Intraperitoneal injection with bovine GH increased plasma IGF-I levels. Four weeks of fasting decreased body weight, increased plasma GH levels, and decreased plasma IGF-I levels. The GH and IGF-I TR-FIAs established herein provide a cost-comparable, non-radioisotopic method for quantifying salmonid plasma GH and IGF-I using commercially available reagents.

Keywords: Fasting; GH; Growth; IGF-I; Immunoassay; Salmon.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animal Feed
  • Animals
  • Cattle
  • Ethanol / pharmacology
  • Fishes
  • Fluoroimmunoassay / instrumentation*
  • Fluoroimmunoassay / methods*
  • Growth Hormone / blood*
  • Insulin-Like Growth Factor I / biosynthesis*
  • Oncorhynchus mykiss / blood*
  • Peptides / chemistry
  • Pituitary Gland / metabolism
  • Salmon / metabolism*

Substances

  • Peptides
  • Ethanol
  • Insulin-Like Growth Factor I
  • Growth Hormone