Validation of an immunochromatographic assay kit based on colored latex particles for the identification of the canine visceral leishmaniasis.

Visceral leishmaniasis is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies. The dogs are main reservoir for human infection. A rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. The aim of this study was to assess the performance of a rapid immunochromatographic strip test based on functionalized colored particles and a new recombinant antigenic protein, as a visual "in situ" method for the diagnosis of canine visceral leishmaniasis. The results were evaluated using an in-house ELISA assay with the same antigen. Both tests produced concordant results and the immunochromatographic strip test showed good diagnostic sensitivity (98%) and specificity (95%). Finally, meta-analysis was used to compare the sensitivity and specificity of the here developed test with the results of commercial immunochromatographic strip tests obtained from literature.

A lateral flow immunoassay based on colored latex particles for detection of canine visceral leishmaniasis.

Canine visceral leishmaniasis (CVL) is the major source of human visceral leishmaniasis. To control the spread of this disease, early and accurate detection of infected dogs is critical but challenging. The serological diagnosis of CVL remains problematic because there are no reliable commercially available tests. Most laboratories use enzyme-linked immunosorbent assay or the indirect immunofluorescent antibody test. These tests use Leishmania chagasi recombinant antigens K39 or K26 assembled with either gold-labelled Staphylococcus aureus protein A or protein G from Streptococcus pyogenes. In this work, we propose the development, optimization and standardization of a lateral flow immunoassay (LFIA) based on functionalized colored particles and a specific recombinant antigen, as a visual in situ method for the diagnosis of CVL. The following analysis variables were considered: (i) the concentration of the latex-protein complex; (ii) the dilution of the serum; (iii) the composition of the employed buffers; (iv) the nominal capillary flow time through the nitrocellulose membrane; (v) the concentration of reagents fixed in the test and control lines; (vi) the particle size of the colored latex; and (vii) the conjugation method. Then, the obtained strips were evaluated as a visual diagnostic tool based on a panel of positive and negative sera. It was observed that because of its simplicity and performance the LFIA test is a quick and reliable alternative for the diagnosis of CVL either in conventional laboratories or for remote areas where laboratories are not readily accessible for conventional assays.

Effect of Delivery Platforms Structure on the Epidermal Antigen Transport for Topical Vaccination.

Transdermal immunization is highly attractive because of the skin's accessibility and unique immunological characteristics. However, it remains a relatively unexplored route of administration because of the great difficulty of transporting antigens past the outermost layer of skin, the stratum corneum. In this article, the abilities of three poly( N-vinylcaprolactam) (PVCL)-based thermoresponsive assemblies-PVCL hydrogels and nanogels plus novel film forming PVCL/acrylic nanogels-to act as protein delivery systems were investigated. Similar thermal responses were observed in all systems, with transition temperatures close to 32 °C, close to that of the skin surface. The investigated dermal delivery systems showed no evidence of cytotoxicity in human fibroblasts and were able to load and release ovalbumin (OVA), a well-studied antigen, in a temperature-dependent manner in vitro. The penetration of OVA into ex vivo human skin following topical application was evaluated, where enhanced skin delivery was seen for the OVA-loaded PVCL systems relative to administration of the protein alone. The distinct protein release and skin penetration profiles observed for the different PVCL assemblies were here discussed on the basis of their structural differences.

Development of a simple and economical diagnostic test for canine leishmaniasis.

Visceral leishmaniasis is a public health problem worldwide. The early diagnosis in dogs is crucial, since they are an epidemiologically relevant reservoir of the disease. The aim of a field study is to early identify the disease allowing rapid intervention to reduce its effects. We propose an immunoagglutination test as a visual in situ method for diagnosis of canine visceral leishmaniasis. Latex-protein complexes were sensitized by covalent coupling of a chimeric recombinant antigen of Leishmania spp. onto polystyrene latex with carboxyl functionality. The reaction time and the antigen concentration under which the immunoagglutination assay shows greater discrimination between the responses of a positive control serum and a negative control serum were determined. Then, the latex-protein complexes were evaluated as a visual diagnostic tool with a panel of 170 sera. The test may be read between 2 and 5 min and can be performed even using sera with elevated concentration of lipids, bilirubin or with variable percentage of hemolysis. The sensitivity, the specificity and the diagnostic accuracy were 78%; 100% and >80%, respectively. The visual immunoagglutination test is of potential application as a method for field studies because it shows results in less than 5 min, it is easy to implement and does not require sophisticated equipment.

Diagnosis of toxoplasmosis in pregnancy. Evaluation of latex-protein complexes by immnunoagglutination.

The aim of this work was to obtain a reagent based on latex particles for ruling out acute toxoplasmosis in pregnant women by immunoagglutination (IA). Latex-protein complexes (LPC) were previously synthesized coupling the recombinant protein of Toxoplasma gondii P22Ag and the homogenate of the parasite to latex particles with different size, chemical functionality and charge density. LPC were tested in IA assays against a panel of 72 pregnant women serum samples. Results were analysed through receiver operating characteristic curves, determining area under the curve (AUC), sensitivity, specificity positive and negative predictive values (PPV and NPV, respectively). It was observed that the antigenicity of proteins was not affected during sensitization by either physical adsorption or covalent coupling. The best results in the sense of maximizing discrimination of low avidity sera from chronic ones were observed for the IA test based on latex particles with carboxyl functionality and the recombinant P22Ag, obtaining an AUC of 0·94, a sensitivity of 100% and a NPV of 100%. In this way, the proposed test could be useful for the toxoplasmosis diagnosis in pregnant women, with the advantages of being cheap, rapid and easy to be implemented.

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

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.

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

OBJECTIVE: 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. METHODS: Complexes' behaviour using total parasite homogenate (TPH), two simple recombinant proteins (RP1 and RP5) and two chimeric recombinant proteins (CP1 and CP2) was comparatively evaluated. The area under ROC curves was used as an index of accuracy. Sensitivity, specificity and discrimination efficiency were assessed. RESULTS: All recombinant antigens showed higher specificity than TPH. The lower specificity of TPH was mainly due to cross-reacting peptides between T. cruzi and Leishmania spp. In turn, all performance indicators were higher for CP1 and CP2 than for RP1 and RP5. The carboxylated latex-CP2 (C2-CP2) complex was able to detect antibodies against T. cruzi. The values of area under ROC curve (0.96), sensitivity (92.3%, 95% CI: 79.4-100.0%) and specificity (84.0%, 95% CI: 67.6-100.0%) indicate that the assay could be used as a screening test. CONCLUSION: The C2-CP2 complex could be an important tool to carry out sero-epidemiological studies.

Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.

The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease.

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

OBJECTIVE: 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. METHODS: The following variables were determined: (i) the sensitisation mechanism, (ii) the emulsifier employed for protein desorption, (iii) the reaction time, (iv) the ionic strength of the reaction medium, (v) the particle concentration, (vi) the presence of blocking agents, (vii) the presence of polyethyleneglycol as potentiator of reaction and (viii) the antigen and antibody concentrations. The search of optimal conditions was investigated by varying one variable at a time. To this effect, monodisperse latex particles sensitised with a recombinant chimeric protein (CP1) were subjected to different conditions. The agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. RESULTS: The maximum discrimination between negative and positive sera was obtained at a reaction time of 5 min, when latex complexes with a concentration of covalently coupled protein of 2.90 mg/m(2) were put in contact with undiluted sera in buffer borate pH 8-20 mm containing glycine (0.1 m) and polyethyleneglycol 8000 (3% w/v). Finally, the latex-protein complex was tested under the obtained optimal conditions, with a panel of Trypanosoma cruzi-positive sera, leishmaniasis-positive sera and -negative sera for both parasites. CONCLUSION: The immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. This test is rapid, easy to implement and could be used under field conditions; but its results should be confirmed by reference techniques like ELISA, HAI, and IFI.

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.

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.

Latex of immunodiagnosis for detecting the Chagas disease. I. Synthesis of the base carboxylated latex.

This article investigates the synthesis of two (monodisperse, carboxylated, and core-shell) latexes, through a batch and a semibatch emulsion copolymerizations of styrene (St) and methacrylic acid (MAA) onto polystyrene latex seeds. A mathematical model of the process was developed that predicts conversion, average particle size, and surface density of carboxyl groups. The model was adjusted to the batch reaction measurements, and then it was used in the design of the semibatch experiment. The semibatch reaction involved an initial homopolymerization of St followed by instantaneous addition of MAA-St-initiator. Compared with the batch reaction results, the semibatch policy more than doubled the surface density of carboxyl groups. The second part of this series describes the development of an immunodiagnosis latex-protein complex for detecting the Chagas disease, by coupling an antigen of Trypanosoma cruzi onto the produced carboxylated latexes.

Latex of immunodiagnosis for detecting the Chagas disease: II. Chemical coupling of antigen Ag36 onto carboxylated latexes.

A novel immunodiagnosis reagent for detecting the Chagas Disease was developed, by chemical coupling of antigen Ag36 of Trypanosoma cruzi onto two (carboxylated and core-shell) latexes. The coupling reactions involved the use of a carbodiimide intermediate. Bovine serum albumin (BSA) was used as a model protein for determining the appropriate conditions for its physical and chemical coupling. BSA showed an increased adsorption onto the base carboxylated latexes, with respect to a PS latex without carboxyl groups. The chemical bonding experiments only involved the carboxylated latexes. With BSA, the final density of covalently bound protein was 2.30 mg/m(2). In addition, around 55% of the total linked protein was chemically coupled, and the reaction was little affected by the pH. With Ag36, the final density of covalently bound protein was 2.44 mg/m(2), around 80% of the total linked protein was chemically coupled, and the chemical coupling was maximum at pH = 5 (i.e., close to the isoelectric point).

Contamination by larger particles of two almost-uniform latices: analysis by combined dynamic light scattering and turbidimetry.

Multiangle dynamic light scattering (MDLS) and turbidimetry (T) were applied (both individually and combined) for determining the contamination by larger particles of two almost-uniform polystyrene (PS) latices. Latex 1 was synthesized in our laboratories, and it contained a main population diameter of 340 nm together with a small fraction of larger particles. This latex was used as the base material for producing an immunoassay kit. Latex 2 was obtained by a simple blend of two uniform PS standards. The proposed data treatment calculates the diameter and number fraction of the large particles contamination assuming that the PSDs are bimodal. The calculation involves minimizing the errors between the measurements and their theoretical predictions. When analyzed by combined MDLS-T, the contamination of Latex 1 involved number fraction 0.6% and particle diameter 865 nm. The T average diameter is a function of the measurement wavelength, and the highest deviations of this average to an increasing contamination by large particles were always observed at the higher wavelengths. The DLS average diameter is a function of the measurement angle, but in this case it is impossible to determine a priori the angle of observation that provides the largest deviation of this average diameter to an increasing contamination.

Latex particle size distribution by dynamic light scattering: novel data processing for multiangle measurements.

Multiangle dynamic light scattering (DLS) provides a better estimate of particle size distributions (PSD) than single-angle DLS. However, multiangle data treatment requires appropriate weighting of each autocorrelation measurement prior to calculation of the PSD. The weighting coefficients may be directly obtained from (i). the autocorrelation baselines or (ii). independent measurement of the average light intensity by elastic light scattering. However, the propagation of errors associated with such procedures may intolerably corrupt the PSD estimate. In this work, an alternative recursive least-squares calculation is proposed that estimates the weighting coefficients on the basis of the complete autocorrelation measurement. The method was validated through a numerical example that simulates the analysis of a polystyrene latex with a bimodal PSD and with "measurements" taken at 10 detection angles. The ill-conditioned nature of the problem determines that the "true" PSD cannot be recovered, even in the absence of errors. A sensitivity analysis was carried out to determine the effect of errors in the weighting coefficients on the PSD recoveries.