Antibody response in silver catfish (Rhamdia quelen) immunized with a model antigen associated with different adjuvants

Adjuvants are essential to boost the immune response to inoculated antigen and play a central role in vaccine development. In this study, we investigated the efficacy of several adjuvants in the production of anti-bovine serum albumin (BSA) antibodies in silver catfish. Two hundred and seventy juvenile silver catfish (60–80 g) of both sexes were intraperitoneally vaccinated with BSA (200 µg/fish) alone or mixed to the following adjuvants: Freund’s complete adjuvant (FCA), Freund’s incomplete adjuvant (FIA), aluminum hydroxide (AlOH), Montanide, four types of cytosine-phosphate-guanine (CpG) oligodeoxynucleotides (ODNs) and three concentrations of β-glucan, and the immune enhancing property was evaluated by measuring anti-BSA antibodies in blood samples at biweekly intervals. Our results demonstrated that CpGs ODNs and β-glucan were as effective as classical adjuvants (FCA, FIA, AlOH and Montanide) in promoting anti-BSA antibodies and that the kinetics of antibody production induced by all adjuvants used in our study had a similar trend to that observed in other fish species, with a peak at 28 days post-vaccination. These results may be useful for the selection of adjuvants for vaccine formulation intended for silver catfish and for the development of vaccine and vaccination strategies to other fish species.

Serology with ML Flow test in health professionals from three different states of Brazil / Sorologia com o teste ML Flow em profissionais de saude em tres diferentes estados do Brasil

BACKGROUND: In highly endemic countries, transmission and sub-clinical infection of leprosy are likely and the disease manifests itself in individuals without any known close contact with a leprosy patient. Health workers are social contacts belonging to the same network (the Health System) and some of them share the same social environment (nursing assistants) as patients with known patients and / or carriers. OBJECTIVE: To identify ML Flow seropositivity among health professionals. METHODS: We conducted a cross-sectional study using a serological survey with the ML Flow test in 450 health professionals (doctors, nurses and nursing assistants), in order to detect seropositivity in areas of high and low endemicity in municipalities from three Brazilian states (RJ, MS and RS). RESULTS: The results showed general 16% seropositivity, higher in low endemic areas, regardless of whether there was direct care for leprosy patients. Paradoxically, a statistical association was observed between the area studied and seropositivity, as the place with the lowest endemicity (CA) had the highest seropositivity rate (p = 0.033). CONCLUSION: The authors suggest these results are associated with a presence of an unspecified link to bovine serum albumin (BSA), carrier of PGL-1 in the ML Flow test, and recommend expanded seroepidemiological research utilizing tests with human and bovine albumin. .

FUNDAMENTOS: Em países altamente endêmicos a transmissão e infecção sub-clínica da hanseníase provavelmente ocorrem e a doença se manifesta em indivíduos sem qualquer contato próximo conhecido com paciente com hanseníase. Os trabalhadores de saúde são contatos sociais que pertencem à mesma rede (Sistema de Saúde) e alguns deles compartilham o mesmo ambiente social (auxiliares de enfermagem) com pacientes conhecidos e/ou portadores. OBJETIVO: Conhecer a soropositividade ao ML Flow entre os profissionais de saúde. MÉTODOS: Foi realizado um estudo transversal através de inquérito sorológico com o teste ML Flow em 450 profissionais de saúde (médicos, enfermeiros e auxiliares de enfermagem) visando conhecer a soropositividade em áreas de alta e baixa endemicidade em municípios de três estados brasileiros (RS, MS e RJ). RESULTADOS: Os resultados mostraram 16% de soropositividade em geral, mais elevada na área de baixa endemicidade, independente da assistência direta a pacientes com hanseníase. Paradoxalmente foi observada associação estatística entre a área estudada e soropositividade, apontando o lugar de mais baixa endemicidade (CA) com o maior valor (p=0,033). CONCLUSÃO: os autores sugerem a presença de ligação inespecífica a soroalbumina bovina (BSA), carreadora do antígeno PGL-1 no teste ML Flow para explicar os resultados inesperados e recomendam testagem ampliada utilizando testes com albumina humana e bovina. .

Generation and characterisation of monoclonal antibodies specific to Plasmodium falciparum enolase.

BACKGROUND AND OBJECTIVES: Glycolysis is the sole source of energy for the intraerythrocytic stages of Plasmodium falciparum, making glycolytic enzymes putative therapeutic targets. Enolase, a single copy gene in P. falciparum is one such enzyme whose activity is elevated approximately 10-15 fold in infected RBC's. It holds the possibility of having multiple biological functions in the parasite and hence can be a suitable candidate for diagnostic and chemotherapeutic purposes. METHODS: We have aimed at generating parasite-specific reagents in the form of monoclonal antibodies. We have raised monoclonal antibodies against the recombinant P. falciparum enolase. RESULTS: Two IgG monoclonals were obtained with 1:1000 titre and specific for P. falciparum enolase. Apicomplexan parasites including P. falciparum enolase has a plant like pentapeptide sequence (104EWGWS108) which is uniquely different from the host counterpart. A peptide spanning this pentapeptide region (ELDGSKNEWGWSKSK) coupled to BSA was used to raise parasite-specific antibody. Four monoclonals were obtained with 1:1000 titre and of IgM isotype. INTERPRETATION AND CONCLUSION: All the monoclonals are specific for P. falciparum enolase and one of them display reactivity against native P. falciparum enolase signifying this pentapeptide to be surface exposed and immunogenic.

Cambio del isotipo en la respuesta a antígenos T independientes por co-inoculación con antígenos T dependientes / Isotype switch in the response against T independent antigens by co-inoculation with T dependent antigens

Un paradigma clásico de la inmulogía plantea que para que ocurra cambio de isotipo en los anticuerpos es condición sine qua non la presentación del antígeno a un linfócito T colaborador por parte de una célula presentadora de antígenos. En el presente trabajo se diseñó un modelo animal, ratones BALB/c, de respuesta inmune frente a dos antígenos típicos. Se utilizo dextrán como antígeno T independiente (AgTI) y seroalbúmina bovina (SAB) como antígeno T dependiente (AgTD), y se estúdio la respuesta, analizando los isotipos de los anticuerpos específicos producidos. Los resultados obtenidos muestran que la respuesta a dextrán en presencia de SAB ocurre con cambio de isotipo (swith), essencialmente de IgM a IgG. Estos experimentos sugieren que la SAB genera un entorno bioquímico inductor de cambio de isotipo tanto en supropia via de procesamiento como en del dextrán. Los resultados señalan que la asociación exclusiva de los AgTDs con las respuestas em las que ocurre cambio de isotipo es incorrecta. Considerando el modelo propuesto resulta poco probable encontrar in vivo y en forma espontânea casos en los que los AgTIs ingreses al organismo aislados; en cambio, es mucho más probable que el ingreso ocurra conjuntamente con AgTDs, y en consecuencia ocurra cambio de isotipo.

Anticuerpos anti-albúmina bovina (BSA) en niños diabéticos tipo 1 recién diagnosticados y su asociación con lactancia materna y exposición a leche de vaca / Bovine serum albumin (BSA) antibodies in children with recently diagnosed type 1 diabetes with breast feeding and milk exposition

BACKGROUND: Environmental and genetic factors (viruses, toxins and diet) are involved in the aetiology of type 1 diabetes. Among the dietary factors, the role of breast feeding and exposure to cow's milk proteins deserve special attention. AIM: To determine the anti-BSA-IgG levels in type 1 diabetic children and to analyse the possible association with breast feeding duration, exposure to cow's milk and beta pancreatic auto-antibodies. PATIENTS AND METHODS: Blood samples were collected from 161 diabetic children and 144 controls to measure anti-BSA-IgG level, GAD65, IA-2 and ICA autoantibodies. All children answered a questionnaire about dietary habits during infancy. RESULTS: anti-BSA-IgG was positive (using a cut off point of 25.6 ng/ml) in 98 per cent of diabetic children and 0 per cent of the control population. The length of breast feeding or early exposure to cow's milk did not influence the concentration of anti-BSA-IgG. Positive BSA titers did not increase the beta pancreatic reactivity (ICA+, GAD+, IA2+). CONCLUSIONS: Our data confirm the high frequency of anti-BSA-IgG among diabetic children. However, a specific role in the immunological process of type 1 diabetes cannot be attributed to this protein.

Obtención de albúmina bovina por termocoagulación: análisis físicoquímico e inmunohematológico / Obtainment of bovine albumine by thermo-coagulation: physicochemical and immunohemathological analysis

Para la purificación de albúmina bovina al 30 por ciento se realizaron modificaciones al método de termocoagulación desarrollado industrialmente por Schneider y colaboradores en 1975. Se obtuvo un rendimiento de 26 g/l con un 100 por ciento de pureza determinada por electroforesis en geles de poliacrilamida e inmunoelectroforesis y se utilizaron como control tres albúminas comerciales. En el análisis físicoquímico se tuvieron en cuenta los valores de ph, concentración de proteínas, sodio y cloruro de sodio, contenido de pigmentos hemáticos y cantidad de agregados moleculares determinado por cromatografía de filtración en gel. Para los ensayos inmunohematológicos nos guiamos por las normativas internacionales para el uso de la seroalbúmina bovina. En todos los casos la albúmina LABIOFAM cumplió con los controles de calidad establecidos para su uso en el diagnóstico serológico de los Servicios de Transfusión y Bancos de Sangre.

Characterization of specific monoclonal antibodies for detection of mefloquine in body fluids.

Specific monoclonal antibodies (MAbs) to mefloquine conjugated to bovine serum albumin (mefloquine-BSA) were produced by hybridoma technology. The mefloquine-BSA was synthesized by converting mefloquine into hemisuccinate followed by convalently linked to bovine serum albumin (BSA) and coupling with N,N' disuccinimidyl carbonate (DSC). The conjugate was purified by Sephadex G-75 gel filtration using 0.01 M PBS pH 7.2. An average of 19.34 molecules of mefloquine were conjugated to each molecule of protein determined by differential UV absorption spectra of hapten and protein carrier. Sixteen monoclones producing antibody specific to mefloquine were screened by indirect ELISA using homologous antigens. The specificity of MAbs was determined by reacting with BSA and the structurally related antimalarial drug, quinine. Three, three, five and two MAbs belonged to IgG1, IgG2a, IgG2b and IgG3, respectively. Most of the MAbs slightly reacted with quinine-BSA due to the closely related structure of mefloquine to quinine. The selected MAb designated 11F9(G5)G9 which showed no cross reaction with quinine-BSA gave high reactivity with blood samples from malaria patients previously treated with mefloquine when compared to normal blood by indirect ELISA. The preliminary results indicated that such specific MAb could be used as antibody probe for detection of mefloquine in biological fluids.

Partial characterization of serum immunoglobulins of Oreochromis mossambicus.

Serum immunoglobulins of O. mossambicus were purified using chromatography methods--CM affinity gel blue chromatography followed by two step purification involving a combination of ion-exchange and gel filtration chromatography. Studies revealed that O. mossambicus produces only one class of high molecular weight macroglobulin as determined by molecular sieving by Sepharose CL 6-B. Immunoelectrophoresis of purified O. mossambicus serum against rabbit anti O. mossambicus serum gave only a single precipitin line. Further analysis of the immunoglobulin by SDS-PAGE showed that the IgM macroglobulin weighs about 900,000 Da, composed of mu-like heavy chain weighing about 90 kDa each and light chains weighing about 30 kDa each.

Efecto de la combustión del tabaco en la propiedad inmunoquímicas de la albúmina / Effect of tobacco smoke in the immunochemistry properties of albumin

Las condiciones en que se realiza la combustión del tabaco durante el acto de fumar favorecen el arrastre de diferentes componentes del cigarrillo, incluyéndose los que se generan en la ignición y que llegan al aparato respiratorio del sujeto que aspira el humo del tabaco. En el extremo de la combustión del cagarrillo la temperatura alcanza 600ºC pero en el punto opuesto, que se pone en contacto con los labios del fumador, la temperatura es menor; por lo anterior se considera que en el cigarrillo se forma un gradiente de temperatura que puede ser capaz de transportar los productos del tabaco. Para demostrar que sí es posible encontrar componentes inmunorreactivos en el humo del tabaco se diseñó un modelo en el cual se usaron dos series de cigarrillos. La serie experimental de cigarrillos fue inyectada con distintas concentraciones de una solución de albúmina sérica bovina (albúmina sérica bovina) y la control recibió salina-fosfato (SSF) sin ninguna proteína. Ambos grupos de cigarrillos permanecieron a 20ºC durante 48 horas para permitir su secado. Posteriormente, se procedió a la combustión de cada serie por separado y el condensado del humo del tabaco producido se dejó en solución salina-fosfato. Los condensados de humo del tabaco derivados de los cigarrillos con mayor concentración de albúmina sérica bovina presentaron más proteína total que los cigarrillos controles. La identificación de la albúmina sérica bovina, que previamente se había colocado en los cigarrillos, se realizó con técnicas inmunoserológicas en las cuales se demostró una banda correspondiente a la albumina sérica bovina, la cual fue revelada con los anticuerpos policlonales anti-albúmina sérica bovina. Los condensados de humo del tabaco producidos con cigarrillos sin albúmina sérica bovina no reaccionaron con los anticuerpos anti-albúmina sérica bovina. La cuantificación de la albvúmina sérica bovina en cada conjunto de cigarrillos mostró de 15 a 601 mcg por cada cigarrillo, de acuerdo con la dosis de albúmina sérica bovina aplicada inicialmente. Se concluye que una fracción de la proteína puesta a los cigarrillos conserva su inmunorreactividad en el condensado del humo del tabaco; por lo cual es posible que los sujetos fumadores al aspirar el humo se pongan en contacto con estructuras del tabaco que desencadenan una respuesta inmunitaria anti-tabaco

Diel variations in antibody response to bovine serum albumin in Oreochromis mossambicus (Peters).

Influence of time of antigen administration on antibody response to bovine serum albumin in the fish, O. mossambicus and variations in serum antibody titre during a 24 hr cycle are reported. Significant variations were observed in magnitude of antibody response in groups of fishes immunized at different times of the day. Magnitude of the response was maximal in fish immunized at 16 hr and minimal in those immunized at 4 hr. Serum antibody titres in a 24 hr study also varied markedly with general elevation during day time, reaching the maximum at 12 hr and depression during night with the lowest titre at 4 hr. These results can be positively correlated to diel variation in number of leucocytes or proportion of lymphocytes which in turn is possibly correlated (negatively) to the periodicity in plasma corticosteroid level.

Proteínas de la leche y su asociación con diabetes mellitus insulino-dependiente / Milk proteins and it association with insulin dependent diabetes mellitus

Cow`s milk has been implicated as a possible trigger of the autoinmune respose that destroys pancreatic beta cells in genetically susceptible host, thus causing diabetes mellitus. Studies in animals have suggested that bovine serum albumin, BSA, is the milk protein responsible, and an albumin peptide containing 17 amino acids, ABBOS, may be the reactive epitope. Antibodies to this peptide react with p69 a beta cell surface protein that may represent the target antigen for milk-induced beta-cell specific inmunity. The consumption of cow`s milk in Europe may support the hypothesis that cow`s milk may be a triggering factor for the development of type I diabetes. However, several epidemiological studies has not found significance correlation between the observed the importance of several enviromental factors in the devepment of type I diabetes with the objetive to establish a good prevention in susceptible subjects

Filter paper blood spot test for detection of anti-ND-BSA antibodies in school children.

For identifying individuals at 'high risk' for developing leprosy, a simplified technique of collection of blood samples on filter paper for detection of anti -ND-BSA antibodies, was optimised. Anti-ND-BSA antibody reactivity on the filter paper was lost on storage at room temperature, but was stable at least for a period of 8 wk at lower temperature. Among the 1495 children screened, 166 (11.1%) were lepromin negative and 122 (8.2%) positive for anti-ND-BSA antibody. In the first phase, 7 of 871 children followed up for 2 yr developed leprosy, while in the second phase 2 of 624 children followed-up for 9 yr developed leprosy. The positivity and negativity of anti-ND-BSA antibodies and lepromin in these I children indicate that the positive status for anti-ND-BSA antibodies has a better predictive value than negative lepromin reactivity. Measurement of anti-ND-BSA antibodies was also of value for monitoring the efficacy of therapy and course of the disease. Extrapolation of the two tests to the total population of children evaluated showed that an individual with negative lepromin reactivity along with presence of anti-ND-BSA antibodies is at a higher risk for developing leprosy than those who have both the tests normal. Lepromin reactivity alone was not of much value for the prediction for development of the disease. However, the presence of anti-ND-BSA antibody was a better indicator for the development of the disease. The probability of developing leprosy in a child with any one of the tests abnormal was higher as compared to a child having all the tests normal.

Efecto de propiltiuracilo sobre la producción de anticuerpos antiseroalbúmina bovina en ratas / Effect of propylthiouracil the production bovine antiserum albumin antibodies in rat

Se presentan los resultados de un estudio realizado in vivo para evaluar el posible efecto inmunosupresor de pripiltiuracilo (PTU). Se utilizó seroalbúmina bovina (SAB) como inmunógeno, la cual se inyectó e a ratas en una dosis única de 5 mg. El PTU se administró en una dosis de 2,5 mg/24 horas durante 10 días. Los anticuerps se detectaron incubando el suero inmune con SAB-1 125. Se empleó polietilenglicol (10 g/dL) para separar el antígeno marcado libre. No encontramos una influencia significativa del PTU sobre la generación de anticuerpos anti SAB, resultado que no sustenta el efecto inmunosupresor de este antitiroideo. En los últimos años se ha estudiado esta característica de las tionamidas, pero los resultados han sido contradictorios