Validation of Heterologous Radioimmunoassays (RIA) for Growth Hormone (GH) and Insulin-Like Growth Factor (IGF)-I in Phocid, Otariid, and Cetacean Species

Abstract

Growth hormone (GH) is a homeorhetic hormone that coordinates diverse physiological processes to partition nutrients toward lean tissue accretion and milk production, and to inhibit utilization of nutrients by adipose tissue, especially during periods of energy deficiency. Due to age-related declines in GH, GH is negatively correlated with overall growth rate and lipid accretion but positively associated with protein accretion. Circulating GH influences peripheral tissues directly or indirectly and is primarily mediated via an insulin-like growth factor (IGF)-I. Similar to GH, age and nutrition influence IGF-I secretion. In general, IGF-I is positively associated with growth rate and accretion of protein. Since purified pinniped or cetacean GH and IGF-I are not available, heterologous assay techniques must be employed to quantify concentrations of these hormones. Critical to the reliability of heterologous assays are the proper execution of validations to demonstrate the accuracy and precision of the assay for each new species of interest. In this study, we developed and validated heterologous radioimmunoassays for seven marine mammal species using porcine and human antisera for GH and IGF-I quantification, respectively. Sensitivity, recovery of mass, assay precision, parallelism, and dilution linearity were determined for each assay and species. Both assays exhibited excellent parallelism and linearity. Appropriate concentrations of hormone were quantified throughout the range of the standard curve. Importantly, using these assays, we have demonstrated that serum concentrations of GH and IGF-I change with age and nutrient intake in these species. Validation of these assays allows evaluation of metabolic hormones that may provide unique perspectives on physiological regulation of differential growth and nutrient allocation for diverse marine mammal species.

How do overwinter changes in body condition and hormone profiles influenceWeddell seal reproductive success?

“…GH was measured using a porcine RIA with rabbit anti‐porcine antibodies (Barb et al . ; Richmond & Zinn ), and IGF‐1 levels were determined using a bovine RIA with rabbit anti‐human IGF‐1 antisera as the primary antibody (Johnson et al . ; Richmond & Zinn ).…”

Seasonal response of ghrelin, growth hormone, and insulin-like growth factor I in the free-ranging Florida manatee (Trichechus manatus latirostris)

“…Radioactivity of the final pellet was measured using a gamma counter (Wizard Walla 1470, Perkin Elmer, MA). Serum concentrations of GH and IGF-I were determined by heterologous RIA (Richmond and Zinn, 2009). Antisera and standards were purchased from A.F.…”

“…Serially diluted pooled serum from free-ranging West Indian manatees was used to validate heterologous ghrelin, GH, and IGF-I RIA. Standard validation parameters including percent recovery, precision of the assay, parallelism, and dilution linearity were assessed (Richmond and Zinn, 2009). …”

Seasonal influence on the response of the somatotropic axis to nutrient restriction and re‐alimentation in captive Steller sea lions (Eumetopias jubatus)

“…Rabbit-anti-porcine GH antisera (AFP422801Rb) was used with porcine GH standard curve (AFP10864B) to quantify sea lion GH, and IGF-I was quantified using a rabbit-anti-human antibody (AFP4892898) and human IGF-I standard (Lot]01). Detailed methods and validations were published in Richmond and Zinn (2009). Concentrations of IGFBP-2 and IGFBP-3 were determined by Western ligand blot (Freake et al, 2001).…”