Standardization of ELISA for the measurement of Insulin-Like Growth Factor I (IGF-I) in bovine plasm
| Autor: | Maioli, Marcos Antonio [UNESP], Nogueira, Guilherme de Paula [UNESP] |
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| Přispěvatelé: | Universidade Estadual Paulista (Unesp) |
| Jazyk: | portugalština |
| Rok vydání: | 2017 |
| Předmět: | |
| Zdroj: | Scopus Repositório Institucional da UNESP Universidade Estadual Paulista (UNESP) instacron:UNESP |
| ISSN: | 0100-736X |
| Popis: | Made available in DSpace on 2018-12-11T17:36:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01. Added 1 bitstream(s) on 2021-07-15T14:34:22Z : No. of bitstreams: 1 S0100-736X2017001201545.pdf: 1212682 bytes, checksum: fcf271ac951c98a04c92b3047475d015 (MD5) This study aimed to standardize an in house competitive enzyme-linked immunosorbent assay (cELISA) to determine plasma concentrations of total insulin-like growth factor I (IGF-I) for the bovine specie using the amplification biotin-streptavidin peroxidase system. The IGF-I was extracted from insulin-like growth factor binding proteins (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 with a prior incubation of samples with the anti-h-IGF-I antibody and another without previous incubation (simultaneous addition of IGF-I and biotinylated sample). The best results were obtained using the method without the prior incubation, using the following combination of reagents: microplates were coated with 0.25μg/well of anti- rabbit IgG, the specific antibody at a dilution of 1:250,000 and 0.06ng/well of biotinylated IGF-I. The in house methodology showed sensitivity of 50ng/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 inter-assay 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. Departamento de Apoio a Produção e Saúde Animal Faculdade de Medicina Veterinária Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Rua Clóvis Pestana 793, Dona Amélia Departamento de Apoio a Produção e Saúde Animal Faculdade de Medicina Veterinária Universidade Estadual Paulista Júlio de Mesquita Filho (Unesp), Rua Clóvis Pestana 793, Dona Amélia |
| Databáze: | OpenAIRE |
| Externí odkaz: |
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