Detection of specific antibodies against T. gondii in samples with varying concentrations of IgG by capture assay in protein a and reaction with high sensitivity antigen

• Grant number: 17/14687-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: October 01, 2017
• End date: September 30, 2018
• Field of knowledge: Biological Sciences - Parasitology - Protozoology of Parasites
• Principal Investigator: Heitor Franco de Andrade Junior
• Grantee: Aline Bastos Dias do Passos
• Host Institution: Instituto de Medicina Tropical de São Paulo (IMT). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Antibody detection reactions using antigen adsorbed on the solid phase depend on adequate dilution of the biological sample, such as sera. Some samplings do not allow this precise dilution, such as filter paper collections or cell purification supernatants, resulting in less sensitivity and specificity. This is a problem in experimental models, where the small volume of blood of experimental animals forces its sacrifice and makes multiple collections impossible. Adsorption to the solid phase of a fixed amount of antibody ligand would allow uniformity of the amount of IgG captured, without interference from its source, provided there was a large excess of antibodies or high sensitivity of the assay. There are specific IgG ligands generally used for affinity purifications, such as specific antibodies, Staphylococcus aureus protein A (SPa) or Protein G of Streptococcus sp. SPa binds two IgG molecules through its Fc portion releasing four antigen that recognition Fab sites, which could react with labeled antigens. This determination would allow comparing precisely small samples of material of unknown volume obtained from experimental models of this infection or its vaccines without involving the necessary death of the experimental animal for collection of the serum and allowing an adequate comparison. In this project we intend to standardize the quantitative detection of specific anti-T. gondii antibodies using IgG capture assays in protein A adsorbed on the solid phase to fix the total IgG mass and relative quantification of the specific antibodies by reaction with T. gondii antigen Conjugated to high sensitivity markers, such as the avidin-biotin system, in experimental vaccination models for toxoplasmosis. (AU)