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Research Note: Development of a sandwich ELISA for determining plasma growth hormone concentrations in goose.

ABSTRACT: Growth hormone (GH) is required for normal postnatal development in poultry; however, no immunoassay exists to assess its levels in geese plasma, hindering the study of endocrine regulation in this species. We developed a sandwich ELISA to determine the GH concentrations in the plasma of geese. Recombinant goose GH was produced using a eukaryotic expression system and purified for use as the reference standard in ELISA and the antigen for producing the polyclonal antibodies in rabbits. Rabbit anti-goose GH polyclonal antibody was used to coat the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and catalyze the chromogenic reaction of 3,3,5,5-tetramethylbenzidine and H₂O₂. A sigmoidal curve was fitted to the optical density and the log of the standard GH concentration using the four-parameter logistic model. The sensitivity of the assay was less than 0.156 ng/mL. The intra- and interassay coefficients of variation were less than 9 and 13%, respectively. The response curve of the serially diluted plasma samples from geese exhibited a good parallel relationship with that observed for the reference standards. The assay effectively detected differences in GH concentrations in plasma samples from geese at various physiological stages; thus, it will be useful for future study of their growth and metabolism.

SUBMITTER: Chen R

PROVIDER: S-EPMC8743213 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:This study aimed to investigate whether the developmental changes in glucose metabolism were associated with insulin signaling in the middle and later stages of goose embryos. Serum and liver were sampled on embryonic day 19, 22, 25, 28, and day of hatchment, with 30 eggs at each sampling time point, and 6 replicates of 5 embryos. The embryonic growth traits, serum glucose, hormone levels, and the hepatic mRNA expressions of target genes related to glucose metabolism and insulin signaling were measured at each time point. Relative body weight, relative liver weight, and relative body length decreased linearly and quadratically from embryonic day 19 to day of hatchment, while relative yolk weight decreased linearly from embryonic day 19 to day of hatchment. Serum glucose, insulin, and free triiodothyronine levels increased linearly with increasing incubation time, while no differences were observed in serum glucagon and free thyroxine levels. The hepatic mRNA expression related to glucose catabolism (hexokinase, phosphofructokinase, and pyruvate kinase) and insulin signaling (insulin receptor, insulin receptor substrate protein, Src homology collagen protein, extracellular signal-regulated kinase, and ribosomal protein S6 kinase, 70 ku) increased quadratically from embryonic day 19 to day of hatchment. The expression of citrate synthase and isocitrate dehydrogenase mRNA decreased linearly and quadratically respectively from embryonic day 19 to day of hatchment. Serum glucose levels were positively related to serum insulin (r = 1.00) and free triiodothyronine (r = 0.90) levels, as well as the hepatic mRNA expression of insulin receptor (r = 1.00), insulin receptor substrate protein (r = 0.64), extracellular signal-regulated kinase (r = 0.81), and ribosomal protein S6 kinase, 70 ku (r = 0.81) related to insulin signaling. In conclusion, glucose catabolism was enhanced and had positive correlations with the insulin signaling in the middle and later stages of geese embryogenesis.

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Research Note: Development of a sandwich ELISA for determining plasma growth hormone concentrations in goose.

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