Valeria Soledad Garcia

Immunodiagnosis of Chagas disease: Synthesis of three latex-protein complexes containing different antigens of Trypanosoma cruzi

This article describes the physical adsorption and the chemical coupling of 3 antigenic proteins of Trypanosoma cruzi onto polystyrene (PS) based latexes to be used as novel immunodiagnosis reagents for detecting the Chagas disease. The coupled proteins were a homogenate of T. cruzi, or a recombinant protein (either Ag36 or CP1). With the homogenate, between 30 and 60% of the total-linked protein was chemically coupled, showing a small dependence with the pH. For Ag36 and CP1, around 90% of the total-linked protein was chemically coupled, with a maximum coupling at pH 5 (i.e., close to the isoelectric point). The chemical coupling of CP1 was less affected by the pH than the coupling of Ag36.

Synthesis of latex-antigen complexes from single and multiepitope recombinant proteins. Application in immunoagglutination assays for the diagnosis of Trypanosoma cruzi infection.

The physical adsorption and the chemical coupling of recombinant proteins of Trypanosoma cruzi onto polystyrene and core-shell carboxylated particles were respectively investigated with the ultimate aim of producing latex-protein complexes to be used in an immunoagglutination assay able to detect the Chagas disease. To this effect, two single proteins (RP1 and RP5) and a multiepitope protein derived from three antigenic peptides (CP2) were evaluated, and sensitizations were carried out at different pHs. The maximum physical adsorption was produced at pHs close to the protein isoelectric point (i.e., pH 6 for RP5 and pH 5 for RP1 and CP2). High fractions of antigens were chemically bound to the carboxyl groups, and the highest surface density of linked protein was also observed at pHs close to the protein isoelectric point. The three latex-protein complexes obtained by covalent coupling at such pHs were tested with sera from a panel of 16 infected and 16 non-infected patients. In the immunoagglutination assays, the latex-CP2 complex produced the best discrimination between positive and negative sera.

Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes.

To determine the conditions under which the immunoagglutination assay to detect Chagas disease, obtained from a novel latex‐(chimeric recombinant antigen) complex, shows greater discrimination between the responses of a positive control serum and a negative control serum.

Immunoagglutination test to diagnose Chagas disease: comparison of different latex-antigen complexes.

To evaluate the diagnostic performance of novel latex–protein complexes obtained from different antigens of Trypanosoma cruzi through immunoagglutination test using a panel of T. cruzi‐positive sera, leishmaniasis‐positive sera and negative sera for both parasites.

P35 and P22 Toxoplasma gondii antigens abbreviate regions to diagnose acquired toxoplasmosis during pregnancy: toward single-sample assays

Abstract Background: P35 and P22 Toxoplasma gondii proteins are recognized by specific IgG at the early infection stage, making them ideal for acute toxoplasmosis pregnancy control. Both proteins have been studied to discriminate between acute and chronic toxoplasmosis. However, results were hardly comparable because different protein obtainment procedures led to different antigens, the reference panels used were not optimally typified, and avidity tests were either not performed or narrowly examined. Methods: We bioinformatically predicted P35 and P22 regions with the highest density of epitopes, and expressed them in pET32/BL21DE3 alternative expression system, obtaining the soluble proteins rP35a and rP22a. We assessed their diagnostic performance using pregnant woman serum samples typified as: not infected, NI (IgG−, IgM−), typical-chronic, TC (IgM−, IgG+), presumably acute, A (IgG+, IgM+, low-avidity IgG), and recently chronic, RC (IgG+, IgM+, high-avidity IgG). Results: rP35a performed better than rP22a to differentiate A from RC, the areas under the curve (AUC) being 0.911 and 0.818, respectively. They, however, performed similarly to differentiate A from TC+RC (AUC: 0.915 and 0.907, respectively). rP35a and rP22a evaluation by avidity ELISA to discriminate A from RC rendered AUC values of 0.974 and 0.921, respectively. The indirect ELISA and avidity ELISA results analyzed in tandem were consistent with those obtained using commercial kits. Conclusions: rP35a and rP22a features suggest that, with complementary use, they could replace parasite lysate for toxoplasmosis infection screening and for acute toxoplasmosis diagnosis. Our proposal should be validated by a longitudinal study and may lead to a reliable toxoplasmosis pregnancy control, performing tests in only one serum sample.

An improved approach to estimate the avidity index of immunoglobulins: Evaluation of the method using IgG anti- Toxoplasma gondii

Fil: Garcia, Valeria Soledad. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Santa Fe. Instituto de Desarrollo Tecnologico para la Industria Quimica. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnologico para la Industria Quimica; Argentina

Latex particles sensitized with proteins of Leptospira interrogans for application in immunoagglutination assays

ABSTRACT Physical adsorption and chemical coupling of recombinant proteins of Leptospira interrogans onto polystyrene and core-shell carboxylated particles were, respectively, investigated with the aim of producing latex–protein complexes to be used in immunoagglutination assays able to detect the leptospirosis disease in either humans or animals. To this effect, a protein lysate of crude extracts was evaluated, and sensitizations were carried out at different pHs, with the antigenic proteins approach to the particle surface favored at pH close to their isoelectric point. In the covalent coupling experiments, high fractions of proteins were chemically bound to carboxyl groups on the particle surface and higher densities of linked proteins were obtained for particles exhibiting greater carboxyl group densities. The produced latex–protein complexes were tested in immunoagglutination assays, by turbidimetry and a visual method, from a panel that included positive and negative bovine, canine, and human sera. The area under receiver operating characteristic curves was used as an index of accuracy. The complexes obtained by covalent coupling of proteins on the latex of higher density of carboxyl groups allowed an acceptable discrimination between the studied positive and negative sera. GRAPHICAL ABSTRACT