Immunochemical characterization of Glycine max L. Merr. var Raiden, as a possible hypoallergenic substitute for cow's milk-allergic patients.

BACKGROUND: Cows' milk allergy (CMA) is the most common cause of food allergy in infancy. The only proven treatment is the complete elimination of cows' milk proteins (CMPs) from the diet by means of hypoallergenic formulas. Soybean-based formulae are widely used although intolerance to soy has been reported to occur in 15-40% of infants with CMA. OBJECTIVE: The aim of this work was to analyse the in vitro reactivity of the soybean cultivar Raiden, which naturally lacks glycinin A(4)A(5)B(3), to evaluate whether this genotype could be a safe CMP substitute for CMA patients. METHODS: The reactivity of conventional soybean (CS) and Raiden soybean (RS) genotypes and also recombinant glycinin A(4)A(5)B(3) and alphabeta-conglycinin with casein-specific monoclonal antibodies and CMP-specific polyclonal serum was evaluated by immunoblotting and ELISA. A sequential competitive ELISA with the polyclonal antiserum and different soluble inhibitors was performed. In addition, an indirect ELISA with sera of atopic children with CMA was carried out to analyse the IgE-binding capacity of the different soybean components. RESULTS: We have shown that CS contains four components that cross-react with CMP, while RS has only one. The remaining cross-reactive component in RS was identified as alpha-subunit beta-conglycinin. By means of inhibitory ELISA, we demonstrated that CS, RS and the alpha-subunit beta-conglycinin extracts inhibited the binding of CMP-specific antibodies to the CMP-coated solid phase. Finally, we showed that CS, RS and the recombinant proteins were recognized by human CMP-specific IgE antibodies. CONCLUSION: This work shows that although Raiden has fewer cross-reactive components than conventional soybean, it still has a residual cross-reactive component: the alpha-subunit beta-conglycinin. This reactivity might make this genotype unsuitable to treat CMA and also explains adverse reactions to soybean in CMA infants.

Mutation in the P36 gene of Mycobacterium bovis provokes attenuation of the bacillus in a mouse model.

P36 is a member of a family of secreted proteins distributed throughout the genus Mycobacterium. The central domain of these proteins contains several amino acid PGLTS repeats, which differ considerably between species. P36, also called exported repetitive protein (Erp) in M. tuberculosis, has been shown to be associated with virulence since the disruption of its gene impaired multiplication of both virulent M. tuberculosis and M. bovis BCG in cultured macrophages and immunocompetent mice. In order to demonstrate that P36 is a putative virulence factor of wild-type Mycobacterium bovis we generated a P36 mutant by gene disruption and we evaluated its replication in spleen and lungs of infected mice. In this study, the mutant strain displays low levels of multiplication in mice, indicating that the P36 gene is important for in vivo growth of M. bovis.

Serological prevalence of anti-latex IgE antibodies in unselected blood donors in Argentina.

The prevalence of specific IgE to natural rubber latex proteins in the general population remains uncertain. The purpose of this study was to determine the prevalence of sera containing specific IgE antibodies to latex proteins using immunoenzymatic methods. A population of 500 unselected adult voluntary blood donors was the source of the sera used in this study. Two different immunoenzymatic methods (EAST and CARLA) were used to analyze the presence of specific IgE antibodies. Confirmation assay was carried out by inhibition ELISA and immunoblotting. Sera from healthy nonatopic individuals were also used as control. Two hundred and twenty five sera showed higher than normal total IgE levels. Of those, three presented latex specific IgE antibodies, which could be inhibited in a dose-response manner with the natural rubber latex and glove extracts. Several latex allergens were recognized by the IgE antibodies from these positive sera. This low seroprevalence (0.66%) indicates that latex hypersensitivity is not an important problem in the general population. We believe that prevention of latex exposure is only necessary in high risk groups of patients.

Analysis of oligosaccharides involved in the asymmetrical glycosylation of IgG monoclonal antibodies.

The aim of this study is to identify the oligosaccharide residues involved in the asymmetric glycosylation of immunoglobulins. We have studied two anti-DNP monoclonal antibodies of the IgG1 isotype. Results show both qualitative and quantitative differences in the carbohydrates of both monoclonal antibodies and their fragments F(ab')2, Fab' and Fd. One of the antibodies -112D5-, which appears to be homogeneous in the Scatchard plot, has oligosaccharide residues in the L chain and in the Fd of one of the Fab'. On the other hand, 112B2 mAb, which also appears to be asymmetrically glycosylated, shows the bimodal curve characteristic of antibodies with different combining site affinities.

N-glycanase treatment of F(ab')2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity.

The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab' glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab')2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab')2 fragments.

Engineering of a polymeric bacterial protein as a scaffold for the multiple display of peptides.

Protein assemblies with a high degree of repetitiveness and organization are known to induce strong immune responses. For that reason they have been postulated for the design of subunit vaccines by means of protein engineering. The enzyme lumazine synthase from Brucella spp. (BLS) is highly immunogenic, presumably owing to its homodecameric arrangement and remarkable thermodynamic stability. Structural analysis has shown that it is possible to insert foreign peptides at the ten amino terminus of BLS without disrupting its general folding. These peptides would be displayed to the immune system in a highly symmetric three-dimensional array. In the present work, BLS has been used as a protein carrier of foreign peptides. We have established a modular system to produce chimeric proteins decorated with ten copies of a desired peptide as long as 27 residues and have shown that their folding and stability is similar to that of the wild-type protein. The knowledge about the mechanisms of dissociation and unfolding of BLS allowed the engineering of polyvalent chimeras displaying different predefined peptides on the same molecular scaffold. Moreover, the reassembly of mixtures of chimeras at different steps of the unfolding process was used to control the stoichiometry and spatial arrangement for the simultaneous display of different peptides on BLS. This strategy would be useful for vaccine development and other biomedical applications.

Single-shot plasmid DNA intrasplenic immunization for the production of monoclonal antibodies. Persistent expression of DNA.

Monoclonal antibodies (Mc. Abs.) were generated against a 18-kDa protein from Brucella abortus 48 h and 25 days after a single intrasplenic injection of a DNA plasmid containing the expression vector for the protein. Hybridomas were also obtained from spleens injected 3, 5, and 10 days before fusion. Somatic cell fusion of spleen cells from mice, injected with the plasmid DNA, in saline, with the NS-0 myeloma cell line resulted in Mc. Abs of the IgG and IgM Isotypes. IgG antibodies were of the IgG2b and IgG1 subtype. Hybridoma tissue culture supernatants were strongly positive by ELISA at dilutions of up to 1/1200 and produced intense specific bands in immunoblotting. All these antibodies recognized the native recombinant protein (the screening antigen) and some of them also recognized the heat-denatured recombinant 18-kDa protein. When compared to standard procedures of immunization, as well as to intramuscular or gene gun DNA immunizations, this technique results in very early, time saving, strong Mc Abs. It is common knowledge that in order to generate specific hybridomas; spleen cells from immunized animals have to be fused no later than 5 days after the last boost. The fact that through single-shot intrasplenic immunization (SSI) specific hybridomas are generated 25 days after one single injection indicates that the gene coding the p18 protein is being expressed in the spleen for at least 20 days. We propose that plasmid DNA intrasplenic immunization can be a helpful tool for the production of specific hybridomas. This route of immunization could also be helpful in the further understanding of early events of the immune response to genetic immunization by naked DNA injection.

Removal of LPS from a Brucella cytoplasmic fraction by affinity chromatography with an anti-LPS monoclonal antibody as immunosorbent.

Affinity chromatography on polymyxin B-Sepharose 4B is one of the most commonly used methods for the removal of contaminating lipopolysaccharides (LPS). However, the LPS of Brucella spp. do not bind to polymyxin B. An affinity chromatography method with an anti-O antigen of Brucella LPS monoclonal antibody as immunosorbent was developed. The method produced a 1000-fold reduction in the LPS content of the cytoplasmic fraction of B. abortus. The eluted proteins retained their antigenicity. The method, which uses mild physiological conditions, is simple, effective and reproducible.

Antibodies to poliovirus detected by immunoradiometric assay with a monoclonal antibody.

An immunoradiometric assay (IRMA) for the assay of antibodies to poliovirus antigens is described. Dilutions of the test sera or whole (finger prick) blood samples were incubated with the Poliovirus antigen bound to a solid phase and the specific antibody was detected by the addition of a mouse anti-human IgG monoclonal antibody (McAb), which was itself revealed by iodinated sheep IgG anti-mouse F(ab). We have shown that this technique is suitable for the estimation of IgG anti-poliovirus antibodies induced in children following polio vaccine. The present study shows that SPRIA provides a simple and inexpensive method for serological studies with poliovirus particularly for use in large-scale surveys.

Urban outbreak of a Brucella melitensis infection in an Argentine family: clinical and diagnostic aspects.

An outbreak of Brucella melitensis in a family was studied. From the fourteen family members who ate unpasteurized goat cheese nine became ill. Patients included four females and five males of 8 to 75 years old. In seven of the patients the diagnosis was confirmed by positive blood culture for B. melitensis biovar 1. All the patients were analyzed by standard tube agglutination (STA) and standard tube agglutination with 2-mercaptoethanol (STA-2ME) tests at the time of diagnosis. In six of the patients, ELISA assays were used to assess the humoral immune anti-protein and anti-lipopolysaccharide (LPS) responses. Anti-LPS IgG antibodies were detected in all of the patients. Anti-proteins IgG antibodies were present at significant levels in all the studied patients including the STA-2ME negative ones.

Brucella abortus cytoplasmic proteins used as antigens in an ELISA potentially useful for the diagnosis of canine brucellosis.

A preparation of Brucella abortus cytoplasmic proteins was depleted of lipopolysaccharide (LPS) by immunoadsorption with a monoclonal antibody (BC68) specific for the O antigen of B. abortus smooth LPS. This extract was used as antigen in an indirect ELISA for measuring antiprotein humoral immune response in dogs suffering from brucellosis and in healthy controls. All of the affected dogs studied showed IgG antiprotein response, while 2% (2 of 103) of the healthy dogs used as controls gave a positive result. All sera found to be positive by ELISA were also positive by the rapid slide agglutination test. These preliminary results show that the ELISA using B. abortus cytoplasmic proteins could be useful for the specific diagnosis of canine brucellosis.

Diagnosis of canine brucellosis by detection of serum antibodies against an 18 kDa cytoplasmic protein of Brucella spp.

An antigenic capture ELISA was developed to measure serum antibodies against an 18 kDa cytoplasmic protein of Brucella. This assay was used to detect anti-18 kDa reactivity in sera from 30 dogs having confirmed or suspected brucellosis. Antibodies against the 18 kDa protein were found in 26 of them, which were also positive by the slide agglutination test (2ME-RSAT). The overall correlation (positive and negative results) between the ELISA and 2ME-RSAT tests was 93.3%. Additionally, these sera were assayed by an indirect ELISA using a whole extract of cytoplasmic proteins of B. abortus (LPS-free CYT). The results of both ELISAs were coincident in 28 of 30 (93.3%) dogs having confirmed or suspected brucellosis. When a serological follow-up was performed on some dogs having confirmed brucellosis, antibody titers measured by both ELISAs showed a parallel progression. On the other hand, the capture ELISA showed good specificity, since a positive result was obtained only in 2 of 103 sera from healthy dogs. These preliminary results show that the ELISA for detecting serum antibodies against the 18 kDa cytoplasmic protein of Brucella could be useful for the diagnosis of canine brucellosis. This study also shows that the results obtained with this single protein of Brucella are equivalent to those obtained with the whole extract of cytoplasmic proteins.

Serological crossreactivity between Brucella abortus and Yersinia enterocolitica 0:9 I immunoblot analysis of the antibody response to Brucella protein antigens in bovine brucellosis.

Sera from three groups of Brucella abortus infected cattle were examined in immunoblots with the following antigens: sodium dodecyl sulfate/mercapto ethanol (SDS/ME) extracts of two rought B. abortus strains (45/20 and RB51) and rough B. ovis, smooth lipopolysaccharides (SLPS) from B. abortus strain 99 and Y. enterocolitica 0:9, and a cytoplasmic extract from smooth B. abortus strain 19-S. The sera groups were: (1) 26 sera from animals, experimentally infected with B. abortus strain 544, which were all positive in the conventional brucellosis serological tests; (2) 152 sera from naturally infected cattle herds with varying titres in the conventional brucellosis tests, and (3) 30 sera from naturally infected cattle with varying titres in the conventional brucellosis tests and from which B. abortus was cultured. B. abortus strain 99 and Y. enterocolitica serotype 0:9 SLPS staining showed up frequently in all sera groups and correlated well with the strength in the conventional brucellosis tests, confirming the immunodominance of SLPS in B. abortus infections. Another immunodominant component of 50-80 kDa was found in the rough B. abortus 45/20 antigen preparation but not in the B. abortus RB51 and in the B. ovis cell extracts. This component was also recognised by sera from Y. enterocolitica 0:9 infected cattle and is probably a protein-lipopolysaccharide complex. Although many of the sera from B. abortus infected cattle with high titres in the conventional brucellosis tests showed complex protein staining patterns in blots, no protein bands other than the 50-80 kDa bands were found to be immunodominant.

Analysis of the effects of heat treatment on gliadin immunochemical quantification using a panel of anti-prolamin antibodies.

Antigen-labeled capture enzyme-linked immunosorbent assay with four different anti-gliadin monoclonal antibodies and an anti-gliadin serum and two different sample systems (purified gliadin fractions heat-treated in soluble phase and a model of dough simulating a baking process) were employed to study the effects of heat treatment on gliadin quantification. The analysis of purified gliadins showed that there is no particularly heat stable fraction. Remarkably, omega-gliadin did not present a differential heat stability. Reactivity varied depending on the time-temperature conditions of the treatment, the antibody employed, and the fraction analyzed. Heated dough samples showed an impairment of protein extraction depending on the intensity of the treatment. Capillary electrophoresis analysis of extracts showed that each gliadin group is affected to a different extent; omega-gliadin is less modified. Immunochemical analysis of the heat-treated samples using either of the five antibodies showed a decrease in the quantified gliadin, in concordance to the loss in the extracted proteins. Among the different sources of error in gliadin immunochemical quantification, the impairment in extraction efficiency in heat-treated samples appears as a major drawback to be overcome.

Preparative fractionation of gliadins by electrophoresis at pH 3.1 (A-PAGE).

Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.

Detection of allergens in Aedes albifasciatus mosquito (Diptera: Culicidae) extracts by immunological methods.

Aedes albifasciatus (Diptera: Culicidae) is the most common mosquito species in Argentina and it has been demonstrated to be the vector for some pathogens. The objective of this study was to describe the allergen composition of this mosquito species endemic to Argentina using SDS-PAGE and immunoblotting methods. Sera from mosquito-bite allergic subjects were employed. The protein extracts, obtained from thoraxes containing salivary glands, showed a protein pattern with components of apparent molecular weights ranging from 14 to over 94 kDa. Some of the components could bind IgE in the 16, 20, 30, 36 and 50-67 kDa-zones, whereas the 14 kDa fraction detectable by SDS-PAGE did not behave as an allergen with any positive serum. This protein extract was used to develop in vitro assays to detect the presence of serum-specific IgE against proteins from A. albifasciatus (RAST and ELISA). Thirty-five sera from patients showing local reactions after mosquito bites were tested. The 21 positive sera were from subjects with clinical histories of atopic signs. Through immunoblotting, these sera revealed IgE reactivity against several fractions, mainly in the 16, 20, 30, 36 and 50-67 kDa zones. Comparing the serum IgE reactivity pattern against A. albifasciatus and Aedes aegypti, we observed that the main difference was found in the 14 kDa region where a strong reactivity was seen. The immunoblotting inhibition results indicate that there might be species-unique and species-shared antigens between A. albifasciatus and A. aegypti.

Structural, functional and immunological studies on a polymeric bacterial protein.

The characterization of proteins from Brucella spp, the causative agent of brucellosis, has been the subject of intensive research. We have described an 18-kDa cytoplasmic protein of Brucella abortus and shown the potential usefulness of this protein as an antigen for the serologic diagnosis of brucellosis. The amino acid sequence of the protein showed a low but significant homology with that of lumazine synthases. Lumazine is an intermediate product in bacterial riboflavin biosynthesis. The recombinant form of the 18-kDa protein (expressed in E. coli) folds like the native Brucella protein and has lumazine-synthase enzymatic activity. Three-dimensional analysis by X-ray crystallography of the homolog Bacillus subtilis lumazine synthase has revealed that the enzyme forms an icosahedral capsid. Recombinant lumazine synthase from B. abortus was crystallized, diffracted X rays to 2.7-A resolution at room temperature, and the structure successfully solved by molecular replacement procedures. The macromolecular assembly of the enzyme differs from that of the enzyme from B. subtilis. The Brucella enzyme remains pentameric (90 kDa) in its crystallographic form. Nonetheless, the active sites of the two enzymes are virtually identical at the structural level, indicating that inhibitors of these enzymes could be viable pharmaceuticals across a broad species range. We describe the structural reasons for the differences in their quaternary arrangement and also discuss the potential use of this protein as a target for the development of acellular vaccines.

Human brucellosis: immunoblotting analysis of three Brucella abortus antigenic fractions allows the detection of components of diagnostic importance.

Results indicating that analysis of the immune humoral response of brucellosis patients by immunoblotting provides useful information for the characterization of antigenic fractions of possible diagnostic importance in human brucellosis are presented. Sera of 90 patients were obtained: 23 suffering from chronic brucellosis, 20 from acute brucellosis and 47 belonging to the group of serologically positive individuals (SPI) without clinical evidence of active infection at the time of examination, and 35 healthy volunteers. They were tested against three antigenic fractions: cytoplasmic (CYT), outer membrane (OM) and inner membrane (IM). These fractions, which include virtually all the bacterial protein components, were prepared from Brucella abortus 1119/3 strain by detergent solubilization, enzymatic digestion and ultracentrifugation. Results obtained with these fractions showed the existence of antigens that permit the detection of brucellosis patients and their differentiation from SPI patients, with very high sensitivity.