[Show abstract][Hide abstract] ABSTRACT: Abstract Diseases such as leishmaniases are an important cause of morbidity and mortality in Brazil and their diagnoses need to be improved. The use of monoclonal antibodies has ensured high specificity to immunodiagnosis. The development of an immunosensor, coupling a monoclonal antibody to a bioelectronic device capable of quickly detecting Leishmania sp. antigens, both qualitatively and quantitatively, is a promising alternative for the diagnosis of leishmaniasis due to its high specificity, low cost, and portability, as compared to conventional methods. The present work was aimed at developing an immunosensor-based assay for detecting Leishmania infantum antigens in tissues of infected hosts. Four hybridomas producing monoclonal antibodies against L. infantum had their specificity confirmed by ELISA. These antibodies were immobilized on a gold surface, covered with a thin film of 2-aminoethanethiol (cysteamine) and glutaraldehyde, blocked with glycine, and placed into contact with extracts of L. infantum-infected and control non-infected hamster spleens. The developed assay was able to detect 1.8 x 104 amastigotes per gram of infected tissue. These results demonstrated that this assay may be useful for quantifying L. infantum amastigotes in organs of experimental animals for studies on pathogenesis and immunity, and is a promising tool for the development of a diagnostic method, based on antigen detection, of human and dog visceral leishmaniases.
Journal of Parasitology 10/2013; 100(1). DOI:10.1645/GE-3052.1 · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immunoenzymatic assays using gold plates and quartz crystal microbalance (QCM) analysis were carried out in order to evaluate chitosan/IgG interaction. Two chitosan solutions (S1 and S2) were prepared with different concentrations of NaOH (0.8% – S1 and 8% – S2). Absorbances 3-fold higher were obtained when chitosan (S2) was used as support when compared with direct IgG adsorption on gold. S1 on gold showed a better stability (at 22 °C, for 72 h) for IgG immobilization when compared with S2. However, S1 was used on QCM analysis and the IgG adsorption led to a non-Sauerbrey response, in which the mass on the electrode surface promote a proportional increase in the crystal resonant frequency. Direct IgG adsorption on gold electrode led to a 14.19% (±2.43) increase in crystal frequency. When S1 was used as a support for IgG, a better immobilization occurred, causing a 24.34% (±0.75) variation in crystal frequency. The structure of chitosan was shown to be efficient for IgG immobilization both in the immunoenzymatic method and in the QCM system.
[Show abstract][Hide abstract] ABSTRACT: A chitosan-modified carbon fiber electrode (CFE) for dengue virus envelope protein (DENV) was developed. Antibodies against DENV were covalently immobilized on the chitosan (CHIT) matrix after activation with sodium periodate. Cyclic voltammetries and scanning electron microscopies analysis were performed to monitor steps involved in the CFE surface modification. Amperometric response of the competitive immunoassays was generated by hydrogen peroxide reaction with the peroxidase conjugated to DENV and 2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as mediator. The immunosensor showed a lower limit of detection for DENV (0.94 ng mL(-1)) than previously described and a linear range from 1.0 to 175 ng mL(-1), in concentration levels clinically relevant for dengue virus diagnosis. The intra- and inter-assay were respectively 5.8% and 3.6%. The unique and simple design of this immunoassay format provides an economical alternative for the manufacture of other sensitive sensors.
[Show abstract][Hide abstract] ABSTRACT: A biomimetic sensor for the determination of dipyrone was prepared by modifying carbon paste with cobalt phthalocyanine (CoPc), and used as an amperometric detector in a flow injection analysis (FIA) system. The results of cyclic voltammetry experiments suggested that CoPc behaved as a biomimetic catalyst in the electrocatalytic oxidation of dipyrone, which involved the transfer of one electron. The optimized FIA procedure employed a flow rate of 1.5 mL min(-1), a 75 μL sample loop, a 0.1 mol L(-1) phosphate buffer carrier solution at pH 7.0 and amperometric detection at a potential of 0.3 V vs. Ag/AgCl. Under these conditions, the proposed method showed a linear response for dipyrone concentrations in the range 5.0 × 10(-6)-6.3 × 10(-3) mol L(-1). Selectivity and interference studies were carried out in order to validate the system for use with pharmaceutical and environmental samples. In addition to being environmentally friendly, the proposed method is a sensitive and selective analytical tool for the determination of dipyrone.
[Show abstract][Hide abstract] ABSTRACT: Aqueous two-phase systems (ATPS) composed of polyethylene glycol (PEG)–citrate have been used for enzyme partitioning studies. The behavior of lactate dehydrogenase (LDH) from bovine heart crude extract was analyzed using a two-level factorial design in which the PEG molar mass and concentration, the citrate concentration were selected as independent variables, while the purification factor, the partition coefficient (K) and the activity yield were selected as responses. The statistical analysis revealed the effect of PEG molar mass on K. LDH exhibited a better partitioning toward PEG-rich phase and the highest K value (1079.81) was obtained with 42% (w/w) PEG 400 and 7.5% (w/w) citrate concentration. PEG molar mass also influenced the purification factor of the enzyme in the top phase. Possibly these ATPS remove inhibitors present in the extract affording higher enzyme yield.
[Show abstract][Hide abstract] ABSTRACT: A ricina é uma proteína bastante tóxica presente nas sementes de mamona que impossibilita o uso da torta de mamona "in natura", como ração. A torta de mamona destoxificada necessita ainda de métodos de análise que garantam a ausência de traços dessa proteína. Objetivou-se, neste trabalho, produzir e avaliar a sensibilidade e especificidade de anticorpos policlonais anti-ricina, para serem empregados como possíveis componentes de métodos sorológicos na detecção de ricina em torta de mamona destoxificada. Foram avaliadas três doses da proteína: 400, 180 e 100 µg cada uma dividida em duas aplicações em coelhos. A primeira dose foi injetada no animal no início do experimento e a segunda após 21 dias. O método de ELISA indicou que as duas doses menores (100 e 180 µg) induziram respostas imunológicas primária e secundária com produção de anticorpos específicos. Enquanto a dose maior (400 µg) de ricina apresentou uma resposta primária com elevação dos títulos de anticorpos, seguida de uma supressão da resposta. Esse perfil é sugestivo de tolerância imunológica. Pela técnica de Western blotting verificou-se que os anticorpos policlonais produzidos são bastante específicos para a ricina, no entanto, por detectarem ricina na forma nativa e desnaturada não são recomendados para o monitoramento de ricina em torta de mamona destoxificada por tratamento térmico.
Ciência e Agrotecnologia 02/2011; 35(1):124-130. DOI:10.1590/S1413-70542011000100015 · 0.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ricin is a very toxic protein found in castor bean plants, making it impossible to use natural castor cake as animal food. The detoxificated castor cake needs to be analyzed by methods that ensure the absence of traces of this protein. This work had the objective to produce and to evaluate the sensitivity and specificity of anti-ricin polyclonal antibodies, to be employed as component of sorologic methods as the ELISA in the detection of ricin in detoxificated castor cake. Three doses of protein, 400, 180 and 100 mu g were evaluated each one injected twice into rabbit, with one half in the begin of the experiment and the other half after 21 days of immunization. The ELISA method indicated that the lower doses (100 c 180 mu g) induced primary and secondary immunological response with production of specific antibodies, while the higher dose of ricin (400 mu g) showed a primary response with increase of the antibody titre, followed of immunological suppression. This profile suggests immunological tolerance. By Western blotting technique it was verified that polyclonal antibodies are too specific to ricin, however, they detected ricin in native and denaturated form and are not recommended for the monitoring of ricin in detoxificated castor bean cake by heat treatment.
Ciência e Agrotecnologia 01/2011; 35(1):124-130. · 0.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An amperometric biosensor was developed for detection of hydrogen peroxide in milk samples. The biosensor was constructed from the immobilization of peroxidase on printed carbon electrode. Optimization parameters were evaluated in order to obtain better performance of the biosensor. The biosensor showed linearity in the range 5.0 to 40.0 µ mol L-1H2O2 in phosphate buffer. Milk samples without dilution reported the detection limit for H2O2 of 0.42 µmol L-1 and quantification of 1.39 µmol L-1.The biosensor could be a sensitive and inexpensive alternative for the detection of H2O2 in adulterated milk samples.