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Standardization and validation of enzyme immunoassay for quantification bovine luteinizing hormone

Grant number: 17/02193-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): April 01, 2017
Effective date (End): March 31, 2018
Field of knowledge:Agronomical Sciences - Veterinary Medicine - Animal Reproduction
Principal researcher:Guilherme de Paula Nogueira
Grantee:Francine Rodrigues Navarro
Home Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil

Abstract

Due to the recognized participation of luteinizing hormone (LH) in the reproductive processes, different methodologies have been developed to quantify this gonadotrophin. The objective of the present study is standardize and validate an enzyme-linked immunosorbent assay (ELISA) for the quantification of bovine LH using the biotin-streptavidin peroxidase amplification system in a competitive assay. For this purpose binding checks will be performed to evaluate the best combination between the concentration of the specific antibody, capture antibody and biotinylated b-LH which result in optical density close to 1.0. After this initial screening, the tests will start with the standard curve to establish the best diluent for the preparation of the standard solutions, the incubation times, temperature and the washes between each step. Afterwards, the methodology will be applied to plasma samples collected from bovines at different stages of development (heifers, pre-pubertal heifers and cows) and the b-LH concentrations of b-LH resulting from the method in development will be compared with those obtained by radioimmunoassay (RIA). Finally, the method will be subjected to validation procedures such as accuracy, precision, sensitivity, lower quantification limit and linearity, these tests will be performed in quadruplicate in 8 independent assays. The linear and non-linear regression calculations of the standard curves and the correlations between the doses obtained by ELISA and RIA will be performed in GraphPad Prism, version 6.0 for Windows. (AU)

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