DEVELOPMENT AND VALIDATION OF ELISA METHODOLOGIES FOR THE MEASUREMENT OF GONADOTROPINS, INSULIN AND GROWTH HORMONE TO BOVINE SPECIE

• Grant number: 16/13200-6
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2016
• End date: May 06, 2019
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal Investigator: Guilherme de Paula Nogueira
• Grantee: Marcos Antonio Maioli
• Host Institution: Faculdade de Medicina Veterinária (FMVA). Universidade Estadual Paulista (UNESP). Campus de Araçatuba. Araçatuba , SP, Brazil

Abstract

This proposal has two principal objectives, one is the standardization and full validation of competitive ELISA methods using biotin-streptavidin amplification system to quantify insulin, growth hormone, the luteinizing and follicle stimulating hormones for the bovine specie. Another objective will be to carry out the analysis of the steps that will be adopted to the validations to identify patterns of adjustments that can be extrapolated to other hormones, to improve efficiency of the validation process. The Initial step consist in the biotinylation of hormones with subsequent two-dimensional binding checks to assess the best combination of concentration of specific antibody, capture antibody, and biotinylated antigen, as well as the incubation times and temperatures. Thereafter will be performed the tests to establish the workflow and its application in the quantification of samples. After standardization, will be performed validation tests which consist in the evaluation of accuracy, precision, sensitivity, lower limit of quantification and linearity. Also, because this proposal is composed by multiple validations tests, will be conducted analysis on parameters as the amount of labeled antigen to be used per well in mol (not mass) and the possible relationship to the molecular weight, the required amount of capture antibody per cm2, the association between the patterns in bound percentage and its effect on the range of the standard curve and the effects on the sensitivity and limits of quantification. This analysis will be conducted because was possible to observe a possible relationship between the dilutions of the reagents used in immunoassays, standardized earlier, that justifies this proposal (FAPESP process 2014/09135-9). Finally, immunizations are performed to produce specific antibodies to bovine insulin in rabbits, besides evaluating the potential use of milk, collected from immunized animals, as a source of antibodies.