Verónica D. G. Gonzalez

Genotyping of Trypanosoma cruzi Sublineage in Human Samples from a North-East Argentina Area by Hybridization with DNA Probes and Specific Polymerase Chain Reaction (PCR)

We have evaluated blood samples of chronic and congenital Trypanosoma cruzi-infected patients from the city of Reconquista located in the northeast of Argentina where no information was previously obtained about the genotype of infecting parasites. Fourteen samples of congenital and 19 chronical patients were analyzed by hybridization with DNA probes of minicircle hypervariable regions (mHVR). In congenital patients, 50% had single infections with TcIId, 7% single infections with TcIIe, 29% mixed infections with TcIId/e, and 7% had mixed infections with TcIId/b and 7% TcIId/b, respectively. In Chronical patients, 52% had single infections with TcIId, 11% single infections with TcIIe, 26% had mixed infections with TcIId/e, and 11% had non-identified genotypes. With these samples, we evaluated the minicircle lineage-specific polymerase chain reaction assay (MLS-PCR), which involves a nested PCR to HVR minicircle sequences and we found a correlation with hybridization probes of 96.4% for TcIId and 54.8% for TcIIe.

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.

Synthesis and characterization of latex-protein complexes from different antigens of Toxoplasma gondii for immunoagglutination assays

ABSTRACT The acute phase recombinant protein of Toxoplasma gondii P22Ag was expressed and purified and the homogenate of the parasite was obtained from an infected mouse. These antigens were used to produce latex-protein complexes (LPC) through physical adsorption and chemical coupling onto different latexes, with the aim of producing immunoagglutination (IA) reagents able to detect recently acquired toxoplasmosis. Polystyrene and “core-shell” latexes where employed, exhibiting varied particle size, functionality (carboxyl or epoxy), and charge density. In sensitization experiments for producing LPC, the recombinant protein showed better coupling efficiency onto the particles surface than the homogenate and this could be explained by the complex mixture of the homogenate, which includes a large number of proteins of different molecular mass, isoelectric points, and hydrophobicity. The synthesized LPC were employed in IA assays. To this effect, the agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. Experiments against control sera were performed to evaluate the performance of various LPC and it was observed that the IA test based on P22Ag and the carboxylated latex of 350 nm of particle diameter allowed a good discrimination between acute sera and chronic/negative ones. The proposed test is cheap, rapid, and easy to implement. GRAPHICAL ABSTRACT

Real-time study of protein adsorption kinetics in porous silicon

This paper presents an optical method for real-time monitoring of protein adsorption using porous silicon self-supported microcavities as a label-free detection platform. The study combines an experimental approach with a physical model for the adsorption process. The proposed model agrees well with experimental observations, and provides information about the kinetics of diffusion and adsorption of proteins within the pores, which will be useful for future experimental designs.

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.

Thermoresponsive nanogels with film-forming ability

Thermoresponsive nanogels (NGs) with film-forming ability could have great potential in delivery system platforms with controlled and targeted release of drugs or proteins, such as in topical treatment. This platform offers the opportunity of combining both the high loading capacity of an NG dispersion and the delivery capability from a film, which could be previously obtained or directly formed onto the surface where the release is required. Therefore, this article investigates the synthesis of NGs based on poly(N-vinylcaprolactam), a thermoresponsive polymer, and poly(butyl acrylate), which promotes particle coalescence and cohesivity (i.e., film forming). The synthesized NGs have low cytotoxicity and are able to form flexible and smooth films, conserving the thermoresponsivity of the particulated system. The obtained films loaded with a model protein show delivery profiles responsive to temperature changes.

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.

Development and assessment of a new cage-like particle adjuvant

To obtain and assess stable cage‐like particles with low surface charge density, which can be prepared using a standardized, economic and scalable method.