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.

Gelatin and Tannic Acid Based Iongels for Muscle Activity Recording and Stimulation Electrodes.

Iongels are soft ionic conducting materials, usually composed of polymer networks swollen with ionic liquids (ILs), which are being investigated for applications ranging from energy to bioelectronics. The employment of iongels in bioelectronic devices such as bioelectrodes or body sensors has been limited by the lack of biocompatibility of the ILs and/or polymer matrices. In this work, we present iongels prepared from solely biocompatible materials: (i) a biobased polymer network containing tannic acid as a cross-linker in a gelatin matrix and (ii) three different biocompatible cholinium carboxylate ionic liquids. The resulting iongels are flexible and elastic with Young's modulus between 11.3 and 28.9 kPa. The morphology of the iongels is based on a dual polymer network system formed by both chemical bonding due to the reaction of the gelatin's amines with the polyphenol units and physical interactions between the tannic acid and the gelatin. These biocompatible iongels presented high ionic conductivity values, from 0.003 and up to 0.015 S·cm-1 at room temperature. Furthermore, they showed excellent performance as a conducting gel in electrodes for electromyography and electrocardiogram recording as well as muscle stimulation.

Film Forming Nanogels for Needle-Free Transdermal Vaccination.

Transcutaneous immunization (TCI) provides a valuable alternative approach to conventional vaccination because of the high accessibility and the exceptional immunological characteristics of the skin, but its application is limited by the low permeability of the stratum corneum. Although nanogels (NGs) have proven to enhance skin penetration of macromolecules with minimum damage, their use in TCI remains almost unexplored. In this context, this article evaluates the performance of novel film-forming NGs (FF-NGs) as TCI. This TCI platform consists of NGs with multilobular morphology that positively combines the properties of cross-linked poly(N-vinylcaprolactam), like thermoresponsiveness and the ability to load and release a cargo, with the film-forming capacity of low Tg lobes. FF-NGs and formed films are characterized at different levels. Formed films show to be able to uniformly load an antigenic protein and release it with a profile depending on the temperature and on their FF-NGs content. In vivo studies have demonstrated that FF-NGs promote the penetration of not only an antigenic protein, but also an adjuvant until the immunocompetent area of skin, generating an adjuvant-dependent specific immune response. Finally, this study provides a successful proof of concept that FF-NGs can be a powerful tool for the transcutaneous release of complex formulations.

Exploiting cyanine dye J-aggregates/monomer equilibrium in hydrophobic protein pockets for efficient multi-step phototherapy: an innovative concept for smart nanotheranostics.

After several decades of development in the field of near-infrared (NIR) dyes for photothermal therapy (PTT), indocyanine green (ICG) still remains the only FDA-approved NIR contrast agent. However, upon NIR light irradiation ICG can react with molecular oxygen to form reactive oxygen species and degrade the ICG core, losing the convenient dye properties. In this work, we introduce a new approach for expanding the application of ICG in nanotheranostics, which relies on the confinement of self-organized J-type aggregates in hydrophobic protein domains acting as monomer depots. Upon the fast photobleaching, while the dye is irradiated, this strategy permits the equilibrium-driven monomer replacement after each irradiation cycle that radically increases the systems' effectivity and applicability. Gadolinium-doped casein micelles were designed to prove this novel concept at the same time as endowing the nanosystems with further magnetic resonance imaging (MRI) ability for dual-modal imaging-guided PTT. By teaching a new trick to a very old dog, the clinical prospect of ICG will undoubtedly be boosted laying the foundation for novel therapeutics. It is anticipated that future research could be expanded to other relevant J-aggregates-forming cyanine dyes or nanocrystal formulations of poorly water-soluble photosensitizers.

Mucoadhesive and responsive nanogels as carriers for sustainable delivery of timolol for glaucoma therapy.

Topical administration to the eye for the treatment of glaucoma is a convenient route because it increases the patient comfort. Timolol can efficiently diminish the intraocular pressure (IOP) of the eye; however the topical application as a solution of timolol maleate (TM) has poor therapeutic index and presents severe side effects. The encapsulation of timolol in nanomaterials has appeared as a technology to increase its residence time in the eye thus achieving a sustained release and consequently diminishing the doses of this drug and their number. The preparation of nanogels (NGs) based on N-isopropylacrylamide (NIPA) and acrylic acid (AAc), easily synthesized by precipitation/dispersion free radical polymerization, is reported in this paper. Such NGs presented excellent dispersability in eye simulated fluid and ideal size for topical application. NGs can load efficiently timolol through ionic interaction, and the in vitro release showed that NGs deliver timolol in a sustained manner. In vivo sustained efficacy of the NGs-timolol nanoformulations was demonstrated in rabbit's glaucoma model, in which the IOP could be diminished and maintained constant for 48 h with only one application. Overall, the synthesized NGs in combination with timolol have potential as drug delivery system for glaucoma therapy.

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.

Thermally self-assembled biodegradable poly(casein-g-N-isopropylacrylamide) unimers and their application in drug delivery for cancer therapy.

In this work, we report the synthesis of graft copolymers based on casein and N-isopropylacrylamide, which can self-assemble into biodegradable micelles of approximately 80 nm at physiological conditions. The obtained copolymers were degraded by trypsin, an enzyme that is overexpressed in several malignant tumors. Moreover, graft copolymers were able to load doxorubicin (Dox) by ionic interaction with the casein component. In vitro release experiments showed that the in situ assembled micelles can maintain the cargo at plasma conditions but release Dox immediately after their exposition at pH 5.0 and trypsin. Cellular uptake and cytotoxicity assays revealed the efficient delivery to the nucleus and antiproliferative efficacy of Dox in the breast cancer cell line MDA231. Both delivery and therapeutic activity were enhanced in presence of trypsin. Overall, the prepared micelles hold a great potential for their utilization as dual responsive trypsin/pH drug delivery system.

Crossing biological barriers with nanogels to improve drug delivery performance.

The current limitations in the use of nanocarriers to treat constantly evolving diseases call for the design of novel and smarter drug delivery systems (DDS). Nanogels (NGs) are three-dimensional crosslinked polymers with dimensions on the nanoscale and with a great potential for use in the biomedical field. Particular interest focuses on their application as DDS to minimize severe toxic effects and increase the therapeutic index of drugs. They have recently gained attention, since they can include responsive modalities within their structure, which enable them to excerpt a therapeutic function on demand. Their bigger sizes and controlled architecture and functionality, when compared to non-crosslinked polymers, make them particularly interesting to explore novel modalities to cross biological barriers. The present review summarizes the most significant developments of NGs as smart carriers, with focus on smart modalities to cross biological barriers such as cellular membrane, tumor stroma, mucose, skin, and blood brain barrier. We discuss the properties of each barrier and highlight the importance that the NG design has on their capability to overcome them and deliver the cargo at the site of action.

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 and assessment of an improved recombinant multiepitope antigen-based immunoassay to diagnose chronic Chagas disease.

The use of chimeric molecules fusing several antigenic determinants is a promising strategy for the development of low-cost, standardized and reliable kits to determine specific antibodies. In this study, we designed and assessed a novel recombinant chimera that complements the performance of our previously developed chimera, CP1 [FRA and SAPA antigens (Ags)], to diagnose chronic Chagas disease. The new chimeric protein, named CP3, is composed of MAP, TcD and TSSAII/V/VI antigenic determinants. We compared the performance of both chimeric Ags using a panel of 67 Trypanosoma cruzi-reactive sera and 67 non-reactive ones. The sensitivity of CP3 vs CP1 was 100 and 90.2%, and specificity was 92.5 and 100%, respectively. The mixture of CP1 + CP3 achieved 100% of sensitivity and specificity. More importantly, an additional subset of 17 sera from patients with discordant results of conventional serological methods was analysed; the CP1 + CP3 mixture allowed us to accurately classify 14 of them with respect to IIF, the usual technique used in most of the reference centres. These results show an improved performance of the CP1 + CP3 mixture in comparison with enzyme-linked immunosorbent assay and indirect haemagglutination commercial assays.

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.

Development and assessment of a new cage-like particle adjuvant.

OBJECTIVES: To obtain and assess stable cage-like particles with low surface charge density, which can be prepared using a standardized, economic and scalable method. METHODS: To form these nanoparticles, the lipid composition and proportion as well the method were modified in relation to cage-like particles previously described elsewhere. Bovine albumin was used to compare ISPA performance with that of other adjuvants in mice and to assess stability. Adjuvant efficacy was analysed using a mouse model of Trypanosoma cruzi infection, which shows protection against an intracellular infection that needs a strong cellular response. KEY FINDINGS: The new particles were better in terms of level, kinetics and profile of humoral responses than Freund Adjuvant, aluminium hydroxide and Montanide TM ISA 206; they also tended to improve ISCOMATRIX™ performance. Particle size and adjuvant performance were conserved during the 6-month period assessed after preparation. In the model of Trypanosoma cruzi infection, mice immunized with ISPA and trans-sialidase developed high protection. CONCLUSIONS: The obtained nanoparticles were stable and outperformed the other assessed adjuvants in joining together the capacity of most adjuvants to enhance the immune response against specific antigen, to reduce the number of doses, to homogenize the response between individuals and to reach a balanced TH1/TH2 response.

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.