A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1∶106 with E. coli O157:H7 and affinity constants ranged from 1.57×1010 to 2.79×1010 L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food

Cellular and molecular changes and immune response in the intestinal mucosa during Trichinella spiralis early infection in rats

Background:: The main targets of the host's immune system in Trichinella spiralis infection are the adult worms (AW), at the gut level, and the migrant or newborn larvae (NBL), at systemic and pulmonary levels. Most of the studies carried out in the gut mucosa have been performed on the Payer's patches and/or the mesenteric lymph nodes but not on the lamina propria, therefore, knowledge on the gut immune response against T. spiralis remains incomplete. Methods: This study aimed at characterizing the early mucosal immune response against T. spiralis, particularly, the events taking place between 1 and 13 dpi. For this purpose, Wistar rats were orally infected with muscle larvae of T. spiralis and the humoral and cellular parameters of the gut immunity were analysed, including the evaluation of the ADCC mechanism exerted by lamina propria cells. Results: A marked inflammation and structural alteration of the mucosa was found. The changes involved an increase in goblet cells, eosinophils and mast cells, and B and T lymphocytes, initially displaying a Th1 profile, characterised by the secretion of IFN-γand IL-12, followed by a polarization towards a Th2 profile, with a marked increase in IgE, IgG1, IL-4, IL-5 and IL-13 levels, which occurred once the infection was established. In addition, the helminthotoxic activity of lamina propria cells demonstrated the role of the intestine as a place of migrant larvae destruction, indicating that not all the NBLs released in the gut will be able to reach the muscles. Conclusions: The characterization of the immune response triggered in the gut mucosa during T. spiralis infection showed that not only an effector mechanism is directed toward the AW but also towards the NBL as a cytotoxic activity was observed against NBL exerted by lamina propria cells.Fil: Saracino, María Priscila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Vila, Cecilia Celeste. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Cohen, Melina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Gentilini, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Falduto, Guido Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Calcagno, Marcela Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Roux, Estela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; ArgentinaFil: Venturiello, Stella Maris. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Malchiodi, Emilio Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

Carbohydrate epitopes are immunodominant at the surface of infectious Neoparamoeba spp.

Amoebic gill disease, the main disease of concern to the salmon industry in Tasmania, is caused by the amoeba, Neoparamoeba spp. Experimental infection can only be induced by exposure to wild-type (WT) parasites isolated from the gills of infected fish, as cultured amoebae are non-infective. To characterize the surface antigens of WT parasites, we produced monoclonal antibodies (mAbs) using subtractive immunization. Mice inoculated with non-infective parasites were treated with cyclophosphamide, to deplete reactive lymphocytes, and then immunized with different antigen preparations from infective parasites. When whole parasites were used for boosting, the percentage of WT unique mAbs was very high (86%) as was the percentage of mAbs specific for carbohydrate epitopes (89%). When deglycosylated membranes were used, the numbers of mAbs specific for non-carbohydrate epitopes did not increase, but the total number of WT unique mAbs was reduced (86-40%). Using an untreated membrane preparation, the total number of mAbs to surface molecules was very high, but all recognized carbohydrate epitopes. The total number of mAbs recognizing carbohydrate epitopes on the surface of the WT parasites was 97%, suggesting that the dominant epitopes on the surface molecules unique to WT parasites are carbohydrate in nature. © 2007 Blackwell Publishing Ltd

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture. © 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved

Characterisation of an immunodominant, high molecular weight glycoprotein on the surface of infectious Neoparamoeba spp., causative agent of amoebic gill disease (AGD) in Atlantic salmon

Amoebic gill disease can be experimentally induced by the exposure of salmonids to Neoparamoeba spp. freshly isolated from infected fish, while cultured amoebae are non-infective. Results from our previous work suggested that one key difference between infectious and non-infectious Neoparamoeba were the highly glycosylated molecules in the glycocalyx. To characterise these surface glycans or glycoproteins we used a monoclonal antibody (mAb 44C12) specific to a surface molecule unique to infective parasites. This mAb recognised a carbohydrate epitope on a high molecular weight antigen (HMWA) that make up 15-19% of the total protein in a soluble extract of infectious parasites. The HMWA consisted of at least four glycoprotein subunits of molecular weight (MW) greater than 150 kDa that form disulfide-linked complexes of MW greater than 600 kDa. Chemical deglycosylation yielded at least four protein bands of approximate MW 46, 34, 28 and 18 kDA. While a similar HMWA complex was present in non-infective parasites, the glycoprotein subunits were of lower MW and exhibited differences in glycosylation. The four glycoproteins subunits recognised by mAb 44C12 were resistant to degradation by PNGase F, PNGase A, O-glycosidase plus β-1, 4-galactosidase, β-N-acetylglucosaminidase and neuraminidase. The major monosaccharides in the HMWA from infectious parasites were rhamnose, fucose, galactose, and mannose while sialic acids were absent. The carbohydrate portion constituted more than 90% of the total weight of the HMWA from infectious Neoparamoeba spp. Preliminary results indicate that immunisation of salmon with HMWA does not lead to protection against challenge infection; rather it may even have an immunosuppressive effect. © 2010 Elsevier Ltd

IgG recognizing 21-24 kDa and 30-33 kDa tachyzoite antigens show maximum avidity maturation during natural and accidental human toxoplasmosis IgG reconhecendo antígenos de taquizoita de 21-24 kDa e 30-33 kDa mostra avidez máxima de maturação na toxoplasmose humana natural e acidental

We describe the avidity maturation of IgGs in human toxoplasmosis using sequential serum samples from accidental and natural infections. In accidental cases, avidity increased continuously throughout infection while naturally infected patients showed a different profile. Twenty-five percent of sera from chronic patients having specific IgM positive results could be appropriately classified using exclusively the avidity test data. To take advantage of the potentiality of this technique, antigens recognized by IgG showing steeper avidity maturation were identified using immunoblot with KSCN elution. Two clusters of antigens, in the ranges of 21-24 kDa and 30-33 kDa, were identified as the ones that fulfill the aforementioned avidity characteristics.<br>Descrevemos a avidez de maturação de IgGs na toxoplasmose humana usando amostras sequenciais de soro de infecções naturais e acidentais. Em casos acidentais, a avidez aumentou continuamente através da infeção enquanto os pacientes naturalmente infectados mostraram perfil diferente. 25% dos soros de pacientes crônicos que possuiam resultados positivos de IgM específica puderam ser classificados apropriadamente usando exclusivamente os dados do teste de avidez. Para se aproveitar a potencialidade desta técnica, antígenos reconhecidos pela IgG mostrando progressiva avidez de maturação foram identificados usando-se Imunoblot com eluição com KSCN. Dois grupos de antígenos, com limites de 21-24 kDa e 30-33 kDa, foram identificados como aqueles que preenchem as características de avidez acima mencionadas