Measurement of insulin-like growth factor I (IGF-I) in bovine plasm by ELISA

Author(s)

Marcos Antonio Maioli

• Document type: Doctoral Thesis
• Press: Araçatuba. 2016-02-17.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina Veterinária. Araçatuba
• Defense date: 2016-02-01
• Advisor: Guilherme de Paula Nogueira

Abstract

This study aimed to standardize an enzyme-linked immunosorbent assay (ELISA) to determine plasma concentrations of total IGF-I using the amplification biotin-streptavidin peroxidase system in a competitive assay. The IGF-I was extracted from IGFBPs using the acidified glycine buffer followed by the pH neutralization with sodium hydroxide. The microplates were coated with anti-rabbit IgG, thereafter the measurements were carried out using two approaches, one without competition (prior incubation of samples with the anti-hIGF-I antibody) and another with competition (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the competitive method, with the following combination of reagents: microplates were coated with 0.25 µg/well of anti-rabbit IgG, the specific antibody at a dilution of 1:250.000 and 0.06 ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of detection limit of 50 ng/ml, a correlation between doses of 0.945 when compared to a commercial method. In addition, after 33 assays (quantification of 1114 samples) the proposed methodology presented a good precision, with interassay variation coefficients of 12.94% and 20.71% for the high and low controls, respectively. Finally, we concluded that ELISA method for the quantification of total IGF-I using the system biotin-streptavidin-peroxidase amplification in a competitive assay is established and is presented as a useful tool for studies aimed at monitoring the IGF-I concentrations. (AU)

• FAPESP's process: 14/09135-9 - Standardization of measurement of insulin-like growth factor I (IGF-I) by radioimmunoassay and monitoring of changes in concentration of hormone during the development of Nellore heifers under different food systems
• Grantee: Marcos Antonio Maioli
• Support Opportunities: Scholarships in Brazil - Doctorate