Generation and characterization of a recombinant chimeric protein (rCpLi) consisting of B-cell epitopes of a dermonecrotic protein from Loxosceles intermedia spider venom.

A chimeric protein was constructed expressing three epitopes of LiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. This species is responsible for a large number of accidents involving spiders in Brazil. We demonstrated that the chimeric protein (rCpLi) generated is atoxic and that antibodies previously developed in rabbits against synthetic epitopes reactive with rCpLi in ELISA and immunoblot assays. The antibody response in rabbits against the rCpLi was evaluated by ELISA and we have detected an antibody response in all immunized animals. Overlapping peptides covering the amino acid sequence of the rCpLi were synthesized on a cellulose membrane, and their recognition by rabbit anti-rCpLi serum assessed. Three different antigenic regions were identified. The percentage of inhibition of the dermonecrotic, hemorrhagic and edematogenic activities caused by the recombinant protein LiD1r in naïve rabbits was assessed by pre-incubation with anti-rCpLi antibodies. Anti-rCpLi induced good dermonecrotic and hemorrhagic protection. The levels of protection were similar to the antiboides anti-LiD1r. In summary, we have developed a polyepitope recombinant chimeric protein capable of inducing multiple responses of neutralizing antibodies in a rabbit model. This engineered protein may be a promising candidate for therapeutic serum development or vaccination.

Padronização de uma técnica de ELISA indireta para o diagnóstico de Fasciolose hepática pela pesquisa de antígenos nas fezes de búfalos (Bubalus bubalis LINNAEUS, 1758) / Standardization of an indirect ELISA test for the demonstration of fasciola hepatica by the antigens research in the feces of water buffaloes (Bubalus bubalis linnaeus, 1758)

Avaliou-se a presença de antígenos de Fasciola hepatica LINNAEUS,1758 nas fezes de búfalos comprovadamente positivos ou comprovadamente negativos por uma técnica indireta de ELISA. Para tanto se utilizou soro hiperimune de coelho (Oryctolagus cuniculus LINNAEUS, 1758) inoculando-se esses animais com um antígeno de secreção/excreção obtido pela incubação de exemplares de F. hepatica em solução tampão seguida de concentração por diálise. Utilizou-se também conjugado anticoelho-peroxidase e substrato cromogênico ortofenilenodiamino (OPD). Para padronização da técnica foram testadas várias diluições de antígeno frente a diversas concentrações de soro hiperimune, variando-se também as concentrações do conjugado e substrato. Utilizando-se as concentrações de reagentes que apresentaram os melhores resultados, estabeleceu-se o ponto de corte para o teste (“cut-off point”), aplicando-o a dois “pools” de extratos de fezes: um de dez búfalos que comprovadamente apresentavam o parasito em seus fígados, e outro de dez búfalos que comprovadamente não o apresentavam. Uma vez determinado o “cut-off point”, o teste foi aplicado a 220 amostras de fezes de búfalos colhidas diretamente da ampola retal dos animais abatidos em abatedouro comercial. Obteve-se uma sensibilidade de 68 e especificidade de 96, sendo que a confirmação da existência ou não da parasitose foi feita por meio de inspeção sanitária “post-mortem” de rotina dos fígados correspondentes. Devido à sobreposição de alguns resultados positivos e negativos nessa última fase, o teste foi aplicado a amostras adicionais de fezes de 20 animais portadores de mais de 20 exemplares de F. hepatica no fígado e de 20 animais comprovadamente sem o parasito, isso verificado mediante exaustiva dissecação dos ductos biliares. A positividade ou negatividade desses animais foi testada adicionalmente por uma modificação da técnica dos quatro tamises metálicos (UENO et al, 1988). Aplicado o teste de ELISA a esses animais, obtev...

An indirect ELISA test evaluated the presence of Fasciola hepatica LINNAEUS, 1758 antigens in the feces of water positive or negative buffaloes. It was standardized using hyper-immune serum from rabbits inoculated with a/an secretion/ excretion antigen obtained from the incubation of F. hepatica in buffered saline concentrated by dialysis. A conjugated anti-rabbit serum–peroxidase and ortophenyle diamine (OPD) chromogen substrate were also used. Different concentrations of antigen and serum, as well as different concentrations of conjugate and substrate were tested to determine the best combination of reagents to establish a “cut-off point” which applied the test to two pools of fecal extracts of ten water buffaloes, one positive and one negative for F.hepatica. The test was then applied to 220 samples of feces collected directly from the rectum of water buffaloes at an abattoir where the animals’ livers were submitted to macroscopic routine sanitary inspection. The test showed a sensibility of 68 and a sensitivity of 96. Because there was overlapping of some positive and negative results in this last stage, the test was applied to the feces of additional 20 positive and 20 negative water buffaloes. These animals were considered positive when a minimum of 20 parasites were found and negative when no parasites were found even after thorough and careful dissection of their bile-ducts. The positivism or negativity of these animals was further attested by a modification of the four-sieve technique (UENO et al, 1988). The ELISA test applied to these animals yielded a sensibility of 95 and a specificity of 100. Since the tested animals came from an area where there is the Eurytrema coelomaticum GIARD ET BILLET, 1892 and with the objective of verifying the possibility of immunological differential diagnosis between the two parasitizes, a preliminary technique of electro-immune-transference (EITB) was developed using hyper-immune rabbit’s serum against F. hepati...

Se evaluó la presencia de antígenos de Fasciola hepatica LINNAEUS, 1758 en los excrementos de búfalos comprobadamente positivos o comprobadamente negativos por una técnica indirecta de ELISA. Para eso se utilizó suero hiperinmune de conejo (Oryctolagus cuniculus LINNAEUS, 1758), inoculándose esos animales con un antígeno de secreción/excreción obtenido por la incubación de ejemplares de F. Hepatica en solución taponada seguida de concentración por diálisis. También se utilizó conjugado anticonejo peroxidase y sustrato cromogénico ortofenilenodiamino (OPD). Para padronización de la técnica fueron testadas varias diluciones de antígeno frente a diversas concentraciones de suero hiperinmune, variándose también las concentraciones del conjugado y sustrato. Utilizándose las concentraciones de reactivos que presentaron los mejores resultados, se estableció el punto de corte para el test (“cut-off-point”) aplicándolo en dos “pools” de extractos de excrementos: uno de diez búfalos que comprobadamente presentaron parásito en sus hígados y otro de diez búfalos que comprobadamente no lo presentaban. Se evaluó la presencia de antígenos de Fasciola hepatica LINNAEUS, 1758 en los excrementos de búfalos comprobadamente positivos o comprobadamente negativos por una técnica indirecta de ELISA. Para eso se utilizó suero hiperinmune de conejo (Oryctolagus cuniculus LINNAEUS, 1758), inoculándose esos animales con un antígeno de secreción/excreción obtenido por la incubación de ejemplares de F. Hepatica en solución taponada seguida de concentración por diálisis. También se utilizó conjugado anticonejo peroxidase y sustrato cromogénico ortofenilenodiamino (OPD). Para padronización de la técnica fueron testadas varias diluciones de antígeno frente a diversas concentraciones de suero hiperinmune, variándose también las concentraciones del conjugado y sustrato...

Development and evaluation of the neutralizing capacity of horse antivenom against the Brazilian spider Loxosceles intermedia.

Spider bites due to Loxosceles intermedia are currently a major public health problem in South Brazil. About 3000 cases are reported annually. Specific treatment for spider bites is provided by the polyvalent anti-arachnidic antiserum produced by Butantan Institute, São Paulo, Brazil by immunizing horses with mixtures of venoms from Tityus serrulatus and T. bahiensis scorpions, as well as Phoneutria nigriventer and L. gaucho spiders. Due to the large amounts of the anti-arachnidic antivenom required and since L. intermedia venom has some biochemical and pharmacological variations, we have produced a specific anti-L. intermedia antivenom. This study shows that horses immunized with crude L. intermedia venom produced IgG antibodies that neutralized the dermonecrotic and lethal activities of the venom. The neutralizing potency of the anti-loxoscelic antivenom was compared with that of the anti-arachnidic antivenom. Our results indicate that both antivenoms were effective in terms of neutralization. However, the anti-loxoscelic antivenom was more efficient than the anti-arachnidic.

ELISA for the detection of venom antigens in experimental and clinical envenoming by Loxosceles intermedia spiders.

Enzyme linked immunosorbent assays (ELISA) were developed to detect antigens from Loxosceles intermedia spider venom. Hyperimmune horse anti-Loxosceles intermedia IgGs were prepared by immunoaffinity chromatography and used to set up a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity to correctly discriminate the circulating antigens in mice that were experimentally inoculated with L. intermedia venom from those inoculated with L. gaucho, L. laeta, and Phoneutria nigriventer spider venoms, Tityus serrulatus scorpion venom and Bothrops jararaca, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.8 ng of venom per assay. The ELISA also detected antigens in the sera of patients envenomed by L. intermedia. Therefore, after standardization for clinical use this ELISA may be a valuable tool for clinicians and epidemiologists.