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1.
Mem. Inst. Oswaldo Cruz ; 116: e200428, 2021. tab, graf
Article in English | LILACS | ID: biblio-1154875

ABSTRACT

BACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.


Subject(s)
Animals , Dogs , Enzyme-Linked Immunosorbent Assay/standards , Enzyme-Linked Immunosorbent Assay/veterinary , Antibodies, Protozoan/blood , Leishmania infantum/immunology , Dog Diseases/diagnosis , Leishmaniasis, Visceral/diagnosis , Recombinant Proteins/immunology , Brazil , Enzyme-Linked Immunosorbent Assay/methods , Serologic Tests , Sensitivity and Specificity , Leishmania infantum/isolation & purification , Leishmaniasis, Visceral/blood , Leishmaniasis, Visceral/veterinary , Antigens, Protozoan/biosynthesis
2.
Rev. bras. parasitol. vet ; 29(3): e005820, 2020. tab, graf
Article in English | LILACS | ID: biblio-1138096

ABSTRACT

Abstract Ehrlichia canis is the main etiological agent of canine monocytic ehrlichiosis (CME), a globally canine infectious disease. In Brazil, CME is considered to be endemic, and its prevalence can reach 65% in some states. The diagnosis of ehrlichiosis is important for treatment and epidemiological purposes. The E. canis TRP36 (Tandem Repeat Protein) protein elicits the earliest acute-phase antibody response observed during the course of the disease. This study aimed to generate the recombinant TRP36 protein from E. canis São Paulo strain and to evaluate its potential as a tool for the serologic diagnosis of CME. The E. canis São Paulo isolate was cultivated in DH82 lineage cells, and its genomic DNA was obtained. The bacterial DNA fragment encoding the entire ORF of TRP36 was cloned into the pBAD/Thio-TOPO vector and transformed into Escherichia coli DH10B competent cells with the trp36-bearing plasmid for protein expression. To evaluate the protein antigenicity, 16 canine serum samples were previously tested (by PCR and the commercial SNAP®4Dx® serological test). The results were in accordance with the SNAP®4Dx® test. Experiments using this recombinant protein as an antigen, targeting the development of a serologic test based on ELISA methodology, are the next step to produce a reliable, affordable and useful diagnostic tool for CME in Brazil.


Resumo Ehrlichia canis é o principal agente etiológico da erliquiose monocítica canina (EMC), uma doença infecciosa canina globalmente dispersa. No Brasil, a EMC é considerada endêmica, e a infecção pode atingir 65% em cães em alguns estados. O diagnóstico de erliquiose é importante para fins de tratamento e epidemiológicos. A proteína TRP36 de E. canis leva a uma resposta humoral com produção de anticorpos em fase aguda, encontrada durante o curso da doença. O objetivo deste estudo foi obter a proteína TRP36 recombinante da amostra São Paulo de E. canis e avaliar seu potencial como ferramenta para o diagnóstico sorológico da CME. O isolado de E. canis São Paulo foi cultivado em células da linhagem DH82 e o DNA genômico foi obtido. O fragmento de DNA bacteriano que codifica toda a ORF de TRP36 foi clonado no vetor pBAD / Thio-TOPO e transformado em células competentes Escherichia coli DH10B, com o plasmídeo portador de trp36 para expressão de proteínas. Para avaliar a antigenicidade da proteína, 16 amostras de soro canino foram previamente analisadas (por PCR e teste sorológico comercial SNAP®4Dx®). Os resultados estavam de acordo com o teste SNAP®4Dx®. Os experimentos que utilizam essa proteína recombinante como antígeno, visando ao desenvolvimento de um teste sorológico baseado no ELISA, são o próximo passo para produzir um teste de diagnóstico confiável, acessível e útil para o diagnóstico da EMC no Brasil.


Subject(s)
Animals , Dogs , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Recombinant Proteins/genetics , Ehrlichiosis/veterinary , Ehrlichia canis/genetics , Dog Diseases/diagnosis , Recombinant Proteins/immunology , Brazil , Serologic Tests/veterinary , Gene Expression , Cell Line , Ehrlichiosis/diagnosis , Escherichia coli/genetics
3.
Braz. j. microbiol ; 49(supl.1): 68-75, 2018. tab, graf
Article in English | LILACS | ID: biblio-974318

ABSTRACT

Abstract Serological testing and culling infected animals are key management practices aiming eradication of bovine leukemia virus infection. Here, we report the development of an indirect ELISA based on BLV recombinant capsid protein (BLVp24r) to detect anti-BLV antibodies in cattle serum. The BLVp24r was expressed in Escherichia coli and purified by affinity chromatography, and then used to set up the ELISA parameters. The Polysorp ® plate coated with 50 ng of antigen/well and bovine serum diluted 1:100 gave the best results during standardization. Using sera from infected and non-infected cattle we set up the cutoff point at 0.320 (OD450 nm) with a sensitivity of 98.5% and specificity of 100.0%. Then, we tested 1.187 serum samples from dairy (736 samples) and beef cattle (451 samples) with unknown status to BLV. We found that 31.1% (229/736) and 9.5% (43/451) of samples amongst dairy and beef cattle, respectively, had IgGs to BLV. The rate of agreement with a commercial competitive ELISA was 84.3% with a κ value of 0.68. Thus, our BLVp24r iELISA is suitable to detect BLV infected animals and should be a useful tool to control BLV infection in cattle.


Subject(s)
Animals , Cattle , Enzyme-Linked Immunosorbent Assay/methods , Serologic Tests/methods , Enzootic Bovine Leukosis/diagnosis , Leukemia Virus, Bovine/immunology , Capsid Proteins/immunology , Antibodies, Viral/blood , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Enzyme-Linked Immunosorbent Assay/instrumentation , Sensitivity and Specificity , Enzootic Bovine Leukosis/blood , Enzootic Bovine Leukosis/virology , Leukemia Virus, Bovine/isolation & purification , Leukemia Virus, Bovine/genetics , Capsid Proteins/analysis , Capsid Proteins/genetics
4.
Rev. bras. parasitol. vet ; 26(1): 60-66, Jan.-Mar. 2017. tab, graf
Article in English | LILACS | ID: biblio-844136

ABSTRACT

Abstract This study evaluated a recombinant aquaporin 1 protein of Rhipicephalus (Boophilus) microplus (RmAQP1) as antigen in a vaccine against R. sanguineus. Five dogs were immunized with RmAQP1 (10 µg) + adjuvant (Montanide) (G1), and five were inoculated with adjuvant only (G2), three times. Twenty-one days after the last immunization, animals of both groups were challenged with R. sanguineus larvae, nymphs and adults, and their biotic potential was compared. Blood samples were collected before each immunization and every 28 days after the last immunization for 10 weeks. Serum antibody titers (IgG) were assessed by ELISA. We observed that: engorgement period of adult females from G1 was 12% shorter than G2; larvae from G1 had 8.7% longer engorgement period than G2 and weighed 7.2% less; nymphs from G1 had 4.5% shorter engorgement period than G2 and weighed 3.6% less; although the antibody titers increased following the second immunization, they rapidly decreased after the third immunization. Results indicated low immunoprotection of RmAQP1 against adult R. sanguineus ticks, and possible efficacy on larvae and nymphs fed on immunized dogs. Further studies should be performed for a full evaluation of the immunoprotection of RmAQP1 against R. sanguineus infestations in dogs.


Resumo Este estudo avaliou a proteína recombinante (aquaporina) do carrapato Rhipicephalus (Boophilus) microplus como antígeno em vacina contra Rhipicephalus sanguineus. Cinco cães foram imunizados com RmAQP1 (10 µg) + adjuvante (G1) e cinco foram inoculados apenas com adjuvante (G2), três vezes. 21 dias após a última imunização todos os animais foram desafiados com larvas, ninfas e adultos de R. sanguineus, e potencial biótico dos carrapatos foi comparado. Amostras de sangue foram coletadas antes de cada imunização e a cada 28 dias após a última imunização, durante 10 semanas. Títulos de anticorpos dos soros dos cães foram avaliados por ELISA. Resultados: o período de ingurgitamento das fêmeas do G1 foi 12% mais curto que o período de ingurgitamento de G2; o período de ingurgitamento das larvas do G1 8,7% foi mais longo e o peso 7,2% menor que no caso de G2; o período de ingurgitamento das ninfas do G1 4,5% foi mais curto e peso 3,6% menor que no caso do G2; aumento dos títulos de anticorpos do G1 após a segunda imunização e declínio após a terceira imunização. Os resultados indicaram baixo potencial de imunoproteção de RmAQP1 contra R. sanguineus adultos, e possível eficácia contra larvas e ninfas, na dose testada. Sugere-se desenvolver novos estudos para melhor avaliação da eficácia de RmAQP1 contra R. sanguineus em cães.


Subject(s)
Animals , Female , Dogs , Tick Infestations/veterinary , Immunoglobulin G/blood , Immunization/veterinary , Rhipicephalus/immunology , Rhipicephalus sanguineus/immunology , Dog Diseases/prevention & control , Aquaporin 1/immunology , Tick Infestations/immunology , Tick Infestations/prevention & control , Recombinant Proteins/immunology , Immunoglobulin G/immunology , Immunization/methods , Dog Diseases/immunology
5.
Rev. bras. parasitol. vet ; 26(1): 67-73, Jan.-Mar. 2017. tab, graf
Article in English | LILACS | ID: biblio-844125

ABSTRACT

Abstract The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.


Resumo O objetivo do presente estudo foi avaliar a eliminação de oocistos de Toxoplasma gondii em gatos imunizados pela via nasal com proteínas ROP2 de T. gondii. Doze gatos sem raça definida (Felis catus) foram divididos em três grupos experimentais G1, G2 e G3 (n = 4). Os animais do G1 receberam 100 μg de proteínas de rROP2 mais 20 μg de Quil-A, G2 recebeu 100 μg de albumina de soro bovino (BSA) junto com 20 μg de Quil-A, e o G3 recebeu apenas solução salina (grupo de controle). Todos os tratamentos foram realizados pela via intranasal nos dias 0, 21, 42 e 63. O desafio foi realizado em todos os grupos no dia 70 com aproximadamente 800 cistos de tecido da cepa ME-49 por via oral. Os animais de todos os grupos tiveram as suas fezes examinadas e o número de oocistos foi determinado durante 20 dias após o desafio. Os animais de G1 eliminaram menos oocistos (86,7%) do que os grupos controles. O ELISA foi utilizado para detectar IgG e IgA anti-rROP2, no entanto, não houve correlação entre o número de eliminhação de oocistos com os níveis de anticorpos IgG ou IgA. No presente trabalho, apesar da menor produção de oocistos no grupo imunizado (G1) em relação aos grupos controles (G2 e G3), não foi possível associar o uso de rROP2 pela via nasal com proteção contra eliminação de oocistos de T. gondii. Para o futuro, a utilização de outras proteínas recombinantes, ou mesmo vacina de DNA, em combinação com rROP2 poderia ser utilizada para tentar melhorar a eficácia deste tipo de vacina.


Subject(s)
Animals , Cats , Cat Diseases/prevention & control , Protozoan Proteins/immunology , Toxoplasmosis, Animal/prevention & control , Protozoan Vaccines/immunology , Membrane Proteins/immunology , Toxoplasma/immunology , Recombinant Proteins/administration & dosage , Recombinant Proteins/immunology , Administration, Intranasal , Antibodies, Protozoan , Cat Diseases/immunology , Protozoan Proteins/administration & dosage , Toxoplasmosis, Animal/immunology , Adjuvants, Immunologic/administration & dosage , Protozoan Vaccines/administration & dosage , Oocysts/immunology , Quillaja Saponins/administration & dosage , Quillaja Saponins/immunology , Membrane Proteins/administration & dosage
6.
Braz. j. microbiol ; 47(2): 394-402, Apr.-June 2016. tab, graf
Article in English | LILACS | ID: lil-780824

ABSTRACT

Abstract Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291—a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.


Subject(s)
Humans , Bacterial Proteins/analysis , Clostridioides difficile/genetics , Clostridium Infections/microbiology , Antibodies, Bacterial/analysis , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Recombinant Proteins/analysis , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Gene Expression , Blotting, Western , Clostridioides difficile/isolation & purification , Clostridioides difficile/immunology , Clostridium Infections/diagnosis , Ribotyping , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology
7.
Article in English | WPRIM | ID: wpr-36487

ABSTRACT

The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.


Subject(s)
Animals , Cell Line , Immune Sera/genetics , Male , Mice , Protozoan Proteins/genetics , Rabbits , Recombinant Proteins/immunology , Sheep , Sodium-Hydrogen Exchangers/genetics , Toxoplasma/genetics , Toxoplasmosis/parasitology
8.
Braz. j. infect. dis ; 19(4): 403-409, July-Aug. 2015. tab, ilus
Article in English | LILACS | ID: lil-759282

ABSTRACT

Human cytomegalovirus is a ubiquitous pathogen that infects the majority of the world's population. After long period of time co-evolving with human being, this pathogen has developed several strategies to evade host immune surveillance. One of the major trick is encoding homologous to those of the host organism or stealing host cellular genes that have significant functions in immune system. To date, we have found several viral immune analogous which include G protein coupled receptor, class I major histocompatibility complex and chemokine. Chemokine is a small group of molecules which is defined by the presence of four cysteines in highly conserved region. The four kinds of chemokines (C, CC, CXC, and CX3C) are classified based on the arrangement of 1 or 2 N-terminal cysteine residues. UL128 protein is one of the analogous that encoded by human cytomegalovirus that has similar amino acid sequences to the human CC chemokine. It has been proved to be one of the essential particles that involved in human cytomegalovirus entry into epithelial/endothelial cells as well as macrophages. It is also the target of potent neutralizing antibodies in human cytomegalovirus-seropositive individuals. We had demonstrated the chemotactic trait of UL128 protein in our previous study. Recombinant UL128 in vitrohas the ability to attract monocytes to the infection region and enhances peripheral blood mononuclear cell proliferation by activating the MAPK/ERK signaling pathway. However, the way that this viral encoded chemokine interacting with peripheral blood mononuclear cells and the detailed mechanism that involving the virus entry into host cells keeps unknown. Here we performed in vitroinvestigation into the effects of UL128 protein on peripheral blood mononuclear cell's activation and receptor binding, which may help us further understand the immunomodulatory function of UL128 protein as well as human cytomegalovirus diffusion mechanism.


Subject(s)
Humans , Chemokines, CC , Cytomegalovirus , Gene Expression Regulation, Viral/genetics , Leukocytes, Mononuclear/virology , Membrane Glycoproteins/immunology , Viral Envelope Proteins/immunology , Cells, Cultured , Cross-Linking Reagents , Chemokines, CC/genetics , Chemokines, CC/immunology , Cytomegalovirus/genetics , Cytomegalovirus/immunology , Receptors, Chemokine/genetics , Recombinant Proteins/immunology
9.
Braz. j. microbiol ; 45(4): 1449-1454, Oct.-Dec. 2014. ilus, tab
Article in English | LILACS | ID: lil-741299

ABSTRACT

The Brazilian Purpuric Fever (BPF) is a systemic disease with many clinical features of meningococcal sepsis and is usually preceded by purulent conjunctivitis. The illness is caused by Haemophilus influenza biogroup aegyptius, which was associated exclusively with conjunctivitis. In this work construction of the las gene, hypothetically responsible for this virulence, were fusioned with ermAM cassette in Neisseria meningitidis virulent strains and had its DNA transfer to non BPF H. influenzae strains. The effect of the las transfer was capable to increase the cytokines TNFα and IL10 expression in Hec-1B cells line infected with these transformed mutants (in eight log scale of folding change RNA expression). This is the first molecular study involving the las transfer to search an elucidation of the pathogenic factors by horizontal intergeneric transfer from meningococci to H. influenzae.


Subject(s)
Humans , Cytokines/biosynthesis , Epithelial Cells/immunology , Epithelial Cells/microbiology , Haemophilus Infections/immunology , Haemophilus influenzae/immunology , Virulence Factors/immunology , Brazil , Cell Line , Cloning, Molecular , Haemophilus Infections/microbiology , Haemophilus Infections/pathology , Haemophilus influenzae/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Transformation, Bacterial , Virulence Factors/genetics
10.
Yonsei Medical Journal ; : 839-852, 2014.
Article in English | WPRIM | ID: wpr-137025

ABSTRACT

Presently, allergy diagnosis and therapy procedures are undergoing a transition phase in which allergen extracts are being step-by-step replaced by molecule-based products. The new developments will allow clinicians to obtain detailed information on sensitization patterns, more accurate interpretation of allergic symptoms, and thus improved patients' management. In this respect, recombinant technology has been applied to develop this new generation of molecule-based allergy products. The use of recombinant allergens allows full validation of identity, quantity, homogeneity, structure, aggregation, solubility, stability, IgE-binding and the biologic potency of the products. In contrast, such parameters are extremely difficult to assay and standardize for extract-based products. In addition to the possibility of bulk production of wild type molecules for diagnostic purposes, recombinant technology opened the possibility of developing safer and more efficacious products for allergy therapy. A number of molecule-based hypoallergenic preparations have already been successfully evaluated in clinical trials, bringing forward the next generation of allergy vaccines. In this contribution, we review the latest developments in allergen characterization, molecule-based allergy diagnosis, and the application of recombinant allergens in therapeutic setups. A comprehensive overview of clinical trials using recombinant allergens as well as synthetic peptides is presented.


Subject(s)
Allergens/immunology , Humans , Hypersensitivity/immunology , Immunotherapy/methods , Recombinant Proteins/immunology
11.
Yonsei Medical Journal ; : 839-852, 2014.
Article in English | WPRIM | ID: wpr-137019

ABSTRACT

Presently, allergy diagnosis and therapy procedures are undergoing a transition phase in which allergen extracts are being step-by-step replaced by molecule-based products. The new developments will allow clinicians to obtain detailed information on sensitization patterns, more accurate interpretation of allergic symptoms, and thus improved patients' management. In this respect, recombinant technology has been applied to develop this new generation of molecule-based allergy products. The use of recombinant allergens allows full validation of identity, quantity, homogeneity, structure, aggregation, solubility, stability, IgE-binding and the biologic potency of the products. In contrast, such parameters are extremely difficult to assay and standardize for extract-based products. In addition to the possibility of bulk production of wild type molecules for diagnostic purposes, recombinant technology opened the possibility of developing safer and more efficacious products for allergy therapy. A number of molecule-based hypoallergenic preparations have already been successfully evaluated in clinical trials, bringing forward the next generation of allergy vaccines. In this contribution, we review the latest developments in allergen characterization, molecule-based allergy diagnosis, and the application of recombinant allergens in therapeutic setups. A comprehensive overview of clinical trials using recombinant allergens as well as synthetic peptides is presented.


Subject(s)
Allergens/immunology , Humans , Hypersensitivity/immunology , Immunotherapy/methods , Recombinant Proteins/immunology
12.
Braz. j. microbiol ; 44(4): 1215-1222, Oct.-Dec. 2013. ilus, graf, tab
Article in English | LILACS | ID: lil-705289

ABSTRACT

The VPl gene of enterovirus 71 (EV71) was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16), A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25%) from CA16infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD).


Subject(s)
Child, Preschool , Female , Humans , Infant , Male , Antibodies, Viral/blood , Capsid Proteins , Enterovirus A, Human/immunology , Hand, Foot and Mouth Disease/diagnosis , Cloning, Molecular , Capsid Proteins/genetics , Capsid Proteins/immunology , Enterovirus A, Human/genetics , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/genetics , Gene Expression , Immunoglobulin G/blood , Immunoglobulin M/blood , Predictive Value of Tests , Recombinant Proteins , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sensitivity and Specificity , Serologic Tests/methods
13.
Invest. clín ; 54(3): 257-269, sep. 2013. ilus
Article in English | LILACS | ID: lil-740324

ABSTRACT

Neuregulins (NRG) are proteins that belong to the family of epidermal growth factors. It is well established that these factors are essential for the development and maintenance of the nervous system. Due to the difficulty of purifying enough quantities of these factors and the lack of specificity from commercially available antibodies, the aim of this work was to produce antibodies against a synthetic peptide capable to detect and identify neuregulin GGFb isoforms. To accomplish this goal, polyclonal antibodies were raised in hens against a synthetic peptide designed from the GGFb1 extracellular sequence. The sequence analysis was made using different epitope-predicting programs. Our results showed that the peptide sequence selected was immunogenic because it was capable of inducing a specific type B immune response in the experimental animal model. These antibodies were also capable of recognizing a recombinant GGF protein and GGF isoforms present in different samples. Our results suggest that the development of immunoglobulin Y (IgY) using synthetic peptides represents, a valuable tool for neuroscience research.


Las Neuregulinas (NRG) son proteínas que pertenecen a la familia de los factores de crecimiento epidermal. Se ha demostrado que estos factores son esenciales para el desarrollo y mantenimiento de la funcionalidad del sistema nervioso. Debido a la dificultad para purificar estas proteínas y la falta de especificidad de los anticuerpos disponibles comercialmente, el objetivo de este trabajo fue producir anticuerpos contra un péptido sintético capaz de detectar e identificar una isoforma de la Neuregulina (GGFb). Para lograr este objetivo, se desarrollaron anticuerpos en gallinas (IgY) contra un péptido sintético diseñado a partir de la secuencia aminoacídica de la región extracelular de GGFb, utilizando programas de predicción de epítopes. Los resultados demuestran que el péptido seleccionado fue immunogénico debido a que estimuló una respuesta inmune específica tipo B en el modelo utilizado. Estos anticuerpos fueron también capaces de reconocer una proteína recombinante e isoformas de GGF presentes en diferentes muestras biológicas. Nuestros resultados demuestran el potencial valor de las inmunoglobulinas Y (IgY) contra péptidos sintéticos como una herramienta de aplicación para la investigación en neurociencia.


Subject(s)
Animals , Female , Rats , Antibodies, Heterophile/immunology , Chickens/immunology , Immunoglobulins/immunology , Neuregulin-1/immunology , Peptide Fragments/immunology , Antibody Specificity , Antibodies, Heterophile/biosynthesis , Antibodies, Heterophile/isolation & purification , Cells, Cultured , Culture Media, Conditioned , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Immunoblotting , Immunoglobulins/biosynthesis , Immunoglobulins/isolation & purification , Neuregulin-1/analysis , Peptide Fragments/chemical synthesis , Protein Isoforms/analysis , Protein Isoforms/immunology , Rats, Sprague-Dawley , Recombinant Proteins/immunology , Schwann Cells/immunology , Schwann Cells/metabolism , Sciatic Nerve/cytology
14.
Rev. cuba. invest. bioméd ; 32(3): 293-301, jul.-sep. 2013.
Article in Spanish | LILACS | ID: lil-705682

ABSTRACT

Objetivo: para determinar la utilidad de herramientas inmunoinformáticas para detectar péptidos que puedan ser inmunodominantes, y evaluar las diferencias entre las respuestas inmunes de los modelos animales empleados en los estudios preclínicos y en los humanos. Métodos: se modeló la respuesta frente a dos proteínas exógenas: la estreptocinasa recombinante y el antígeno de superficie de la hepatitis B. A partir de sus secuencias primarias se emplearon algoritmos para identificar epítopes B y T frente a moléculas HLA de clase I y II (HLA-A*0201, HLA-DRB1*0301 y HLA-DRB1*0701) y los haplotipos murinos H2-Kd y H2-Kk. Se seleccionaron los péptidos de más alta puntuación. Resultados: el algoritmo ABCPred mostró una mejor capacidad de predicción de epítopes B, mientras fue mayor la coincidencia para los programas de modelación de la respuesta T. Los epítopes generados para el haplotipo H2-Kk tuvieron una similitud mayor con los presentados por las moléculas HLA seleccionadas. Conclusiones: se presenta una metodología aplicable al desarrollo de vacunas de subunidades y multiepitópicas, así como para otros fármacos biotecnológicos de naturaleza peptídica, que permite optimizar las etapas preclínicas y clínicas, a muy bajo costo, mínimos requerimientos tecnológicos, utilización óptima de medios, recursos y capital humano disponibles en cualquier institución del sistema nacional de salud


Objective: determine the usefulness of immunoinformatics tools to detect potentially immunodominant peptides, and evaluate the differences between the immune responses provided by the animal models used in preclinical and human studies. Methods: modeling was conducted of the response to two exogenous proteins: recombinant streptokinase and hepatitis B surface antigen. Based on their primary sequences, algorithms were used to identify B and T epitopes against HLA class I and II molecules (HLA-A*0201, HLA-DRB1*0301 and HLA-DRB1*0701), and murine haplotypes H2-Kd and H2-Kk. The highest scoring peptides were chosen. Results: ABCPred algorithm showed a better prediction capacity for B epitopes, whereas coincidence was greater in modeling programs for the T response. The epitopes generated for haplotype H2-Kk had greater similitude with those presented by the HLA molecules selected. Conclusions: a methodology is presented which is applicable to the development of subunit and multiepitope vaccines, as well as other peptidic biotechnological drugs. This methodology allows optimization of the preclinical and clinical phases at a very low cost, with minimal technological requirements, optimal use of media, and resources and human capital available at any institution of the national health system


Subject(s)
Humans , Hepatitis B Antigens/analysis , Recombinant Proteins/immunology , Vaccines, Subunit/immunology
15.
Rev. Soc. Bras. Med. Trop ; 46(1): 97-99, Jan.-Feb. 2013. ilus, tab
Article in English | LILACS | ID: lil-666813

ABSTRACT

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.


Subject(s)
Animals , Mice , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Recombinant Proteins/immunology , Viral Envelope Proteins/immunology , West Nile virus/immunology , Brazil , Enzyme-Linked Immunosorbent Assay
16.
West Indian med. j ; 62(1): 12-20, Jan. 2013. ilus, tab
Article in English | LILACS | ID: biblio-1045581

ABSTRACT

The rationale of this study was to use several immunological assays to investigate the reactivity of immunoglobulin binding protein (IBP) to immunoglobulins from various avian and mammalian species. The IBP studied were Staphylococcal protein A (SpA), Streptococcal protein G (SpG), Peptostreptococcal protein L (SpL) and recombinant protein LA (SpLA). The various immunological techniques used were double immunodiffusion (Ouchterlony technique) that tested positive high protein reactivities, direct and competitive enzyme-linked immunosorbent assays (ELISAs) that tested moderate and low positive protein binding capacities, respectively. In addition to sandwich ELISAs, immunoblot analyses and Ig-purification by SpA-affinity chromatography, which were sensitive tests and helpful in the screening and confirmatory tests were also used. The Ouchterlony technique showed that compared to the other proteins, SpLA had the highest range of reactivity with animal sera and purified immunoglobulins while SpL was least reactive. With the direct ELISA, SpL reacted with the raccoon sera, rabbit IgG and with IgY from bantam hens and pigeons. While with the direct ELISA, SpA reacted with sera from skunk, coyote, raccoon, mule, donkey and human. The sandwich ELISA revealed high reactivity of both SpG and SpLA with mammalian sera titres ranging from 1:32 (raccoon serum) to 1:1024 (mule and donkey sera).These results suggest that IBP can be used for the detection of immunoglobulin using various immunological assays and this is important for the diagnosis of infectious diseases in animal and bird populations studied and in the purification of immunoglobulins.


El fundamento de este estudio radica en el uso de varios ensayos inmunológicos para investigar la reactividad de la proteína de unión de la inmunoglobulina (IBP) frente a las inmunoglobulinas de varias especies aviarias y mamíferas. Las proteínas IBP estudiadas fueron la proteína estafilocócica A (SpA), la proteína estreptocócica G (SpG), la proteína peptoestreptocócica L (SpL), y la proteína recombinante LA (SpLA). Las varias técnicas inmunológicas usadas fueron: la inmunodifusión doble (técnica de Ouchterlony) para examinar las reactividades positivas de la proteína alta; el ensayo por inmunoabsorción ligado a enzimas(ELISA), de tipo directo y competitivo, para examinar la capacidad de realizar uniones positivas de proteína moderada y baja, respectivamente, además del ensayo ELISA 'Sándwich', los análisis inmunoblot, yla purificación de IgG, mediante cromatografía de afinidad, los cuales fueron pruebas sensibles y útiles en el tamizaje y las pruebas de confirmación. La técnica de Ouchterlony mostró que - en comparación con otras proteínas - la SpLA tenía el grado más alto de reactividad con los sueros animales y las inmunoglobulinas purificadas, mientras que la SpL fue la menos reactiva. Con el ELISA directo, la SpL reaccionó con los sueros de mapache, la IgG de conejo, así como con la IgY de palomas y gallinas de Bantam, en tanto con el ELISA directo, la SpA reaccionó con sueros de mofeta, coyote, mapache, mula, asno y seres humanos. ELISA "sándwich" reveló una alta reactividad tanto de SpG como de SpLA, con títulos séricos mamíferos que iban desde 1:32 (suero de mapache) hasta 1:1024 (sueros de mula y de asno). Estos resultados sugieren que la proteína de unión IBP puede usarse en la detección de la inmunoglobulina usando varios ensayos inmunológicos, lo cual es importante para el diagnóstico de enfermedades infecciosas en las poblaciones animales y aviarias bajo estudio, así como para la purificación de inmunoglobulinas.


Subject(s)
Humans , Animals , Bacterial Proteins/immunology , Birds/immunology , Immunoglobulins/biosynthesis , Chromatography, Affinity , Immunoenzyme Techniques/methods , Mammals/immunology , Recombinant Proteins/immunology , Carrier Proteins/immunology , Communicable Diseases/diagnosis
17.
Journal of Infection and Public Health. 2013; 6 (3): 166-172
in English | IMEMR | ID: emr-142717

ABSTRACT

Dialysis patients are more likely than the general population to develop active tuberculosis [TB]. In these patients, the availability of a highly sensitive and specific test to diagnose latent TB will ensure earlier treatment and decreased progression to active disease. In the current study, the Quanti-FERON-TB Gold In-Tube [QFT-G] test was compared with the tuberculin skin test [TST] for the diagnosis of latent tuberculosis infection [LTBI] among 200 hemodialysis patients and 15 confirmed TB disease cases in a tertiary care center in Saudi Arabia. Among the LTBI cases, 26 [13%] were TST positive, and 65 [32.5%] were positive by the QTF-G test, with an overall agreement between the 2 tests of 75.5% [k = 0.34] being observed. Among the confirmed tuberculosis disease cases, none were positive by TST, and 10 [66.7%] were positive by the QTF-G test, resulting in an overall agreement of 33.3% [k = 0]. A comparison between the TST and the QTF-G test was performed based on the sensitivity, specificity, and area under the curve [AUC] obtained for the tests. The QTF-G test was more sensitive and less specific than the TST in predicting the confirmed TB disease cases. When we tested the correspondence of the AUC values between the 2 diagnostic modalities, the obtained p-value was 0.0003. In conclusion, the AUCs of the examined diagnostic modalities are significantly different in predicting LTBI and tuberculosis


Subject(s)
Humans , Male , Female , Tuberculin Test , Renal Dialysis/adverse effects , Mycobacterium tuberculosis/immunology , Bacterial Proteins/immunology , Recombinant Proteins/immunology , Renal Insufficiency/complications , Sensitivity and Specificity
18.
Mem. Inst. Oswaldo Cruz ; 107(supl.1): 90-94, Dec. 2012. ilus, mapas, tab
Article in English | LILACS | ID: lil-659746

ABSTRACT

Leprosy is a slowly evolving disease that occurs mainly in adults. In this study, the Mamaría Village, state of Portuguesa was selected because it had one of the highest prevalence rates (13.25%) of leprosy cases in 1997. Between 1998-2004, 20.2% of the 89 cases registered in this village were less than 15 years old and 61.8% were males. Pau-cibacillary (PB) lesions were the predominant clinical forms identified, although also multibacillary (MB) forms were found. Additionally, 76% of the patients were bacteriologically negative. At the time of diagnosis, 75% of the patients presented with grade 0 disabilities, 23% with grade 1 and 2% with grade 2. Serum samples were collected from 18 PB and 15 MB patients, in addition to 14 family contacts, at the beginning and end of treatment. All the groups were re-evaluated during a three-year period (2008-2011). The proteins used for evaluation were ML0405, ML2331 and LID-1. These mycobacterial proteins were highly specific for Mycobacterium leprae and the IgG responses decreased in both MB and PB patients during multidrug treatment. Our results suggest that these antigens could be used as markers for successful treatment of non-reactional lepromatous patients.


Subject(s)
Adolescent , Adult , Female , Humans , Male , Young Adult , Bacterial Proteins/blood , Immunoglobulin G/blood , Leprosy, Multibacillary/diagnosis , Leprosy, Paucibacillary/diagnosis , Mycobacterium leprae/immunology , Antibodies, Bacterial/blood , Bacterial Proteins/immunology , Leprosy, Multibacillary/epidemiology , Leprosy, Paucibacillary/epidemiology , Recombinant Proteins/blood , Recombinant Proteins/immunology , Venezuela/epidemiology
19.
Mem. Inst. Oswaldo Cruz ; 107(supl.1): 112-123, Dec. 2012. ilus, graf, tab
Article in English | LILACS | ID: lil-659749

ABSTRACT

Silent transmission of Mycobacterium leprae, as evidenced by stable leprosy incidence rates in various countries, remains a health challenge despite the implementation of multidrug therapy worldwide. Therefore, the development of tools for the early diagnosis of M. leprae infection should be emphasised in leprosy research. As part of the continuing effort to identify antigens that have diagnostic potential, unique M. leprae peptides derived from predicted virulence-associated proteins (group IV.A) were identified using advanced genome pattern programs and bioinformatics. Based on human leukocyte antigen (HLA)-binding motifs, we selected 21 peptides that were predicted to be promiscuous HLA-class I T-cell epitopes and eight peptides that were predicted to be HLA-class II restricted T-cell epitopes for field-testing in Brazil, Ethiopia and Nepal. High levels of interferon (IFN)-γ were induced when peripheral blood mononuclear cells (PBMCs) from tuberculoid/borderline tuberculoid leprosy patients located in Brazil and Ethiopia were stimulated with the ML2055 p35 peptide. PBMCs that were isolated from healthy endemic controls living in areas with high leprosy prevalence (EChigh) in Ethiopia also responded to the ML2055 p35 peptide. The Brazilian EChigh group recognised the ML1358 p20 and ML1358 p24 peptides. None of the peptides were recognised by PBMCs from healthy controls living in non-endemic region. In Nepal, mixtures of these peptides induced the production of IFN-γ by the PBMCs of leprosy patients and EChigh. Therefore, the M. leprae virulence-associated peptides identified in this study may be useful for identifying exposure to M. leprae in population with differing HLA polymorphisms.


Subject(s)
Humans , Cytokines/immunology , Epitopes, T-Lymphocyte/immunology , Mycobacterium leprae/pathogenicity , Virulence/immunology , Brazil , Bacterial Proteins/immunology , Computational Biology , Epitope Mapping , Ethiopia , Mycobacterium leprae/immunology , Mycobacterium leprae/isolation & purification , Mycobacterium leprae/virology , Nepal , Peptide Fragments/immunology , Recombinant Proteins/immunology
20.
Rev. bras. parasitol. vet ; 21(3): 254-262, July-Sept. 2012. ilus, tab
Article in English | LILACS | ID: lil-653714

ABSTRACT

The southern cattle fever tick, Rhipicephalus (Boophilus) microplus, is no doubt the most economically important ectoparasite of cattle globally. The inappropriate use of chemical acaricides has driven the evolution of resistance in populations of R. (B.) microplus. Anti-tick vaccines represent a technology that can be combined with acaricides in integrated control programs to mitigate the impact of R. (B.) microplus. The recombinant form of Bm86 antigen from the Campo Grande (rBm86-CG) strain of R. (B.) microplus was produced using the Pichiapastoris expression system to test its ability to immunoprotect cattle against tick infestation. Secretion of rBm86-CG by P. pastoris through the bioprocess reported here simplified purification of the antigen. A specific humoral immune response was detected by ELISA in vaccinated cattle. Immunoblot results revealed that polyclonal antibodies from vaccinated cattle recognized a protein in larval extracts with a molecular weight corresponding to Bm86. The rBm86-CG antigen showed 31% efficacy against the Campo Grande strain of R. (B.) microplus infesting vaccinated cattle. The rBm86-CG is an antigen that could be used in a polyvalent vaccine as part of an integrated program for the control of R. (B.) microplus in the region that includes Mato Grosso do Sul.


O carrapato Rhipicephalus (Boophilus) microplus é, sem dúvidas, o ectoparasito economicamente mais importante para o gado a nível mundial. A utilização inadequada de acaricidas tem impulsionado a evolução da resistência em populações de R. (B.) microplus. Vacinas contra o carrapato representam uma tecnologia que pode ser combinada com acaricidas em programas de controle integrado para diminuir o impacto de R. (B.) microplus. A forma recombinante da Bm86 da cepa Campo Grande (rBm86-CG) de R. (B.) microplus foi produzido utilizando o sistema de expressão em Pichia pastoris para testar sua capacidade de imunoproteção ao gado contra a infestação de carrapatos. A secreção de rBm86-CG em P. pastoris pelo bioprocesso, simplificou a purificação do antígeno. A resposta imune humoral específica foi detectada por ELISA em soros de bovinos vacinados. Resultados de "imunoblot" revelaram que anticorpos policlonais de bovinos vacinados reconheceram uma proteína em extratos de larvas com um peso molecular correspondente à Bm86. O antígeno rBm86-CG mostrou eficácia de 31% contra a amostra CG de R. (B.) microplus utilizada para infestar os bovinos vacinados. Pelos resultados obtidos, concluímos que a rBm86-CG é um antígeno que pode ser usado em uma vacina polivalente, como parte de um programa integrado para o controle de R. (B.) microplus no estado do Mato Grosso do Sul, Brasil.


Subject(s)
Animals , Cattle , Female , Male , Cattle Diseases/parasitology , Cattle Diseases/prevention & control , Membrane Glycoproteins/immunology , Membrane Glycoproteins/therapeutic use , Recombinant Proteins/immunology , Recombinant Proteins/therapeutic use , Rhipicephalus/immunology , Tick Infestations/veterinary , Vaccines/immunology , Tick Infestations/prevention & control , Vaccines/therapeutic use
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