RESUMO
The electronegative low-density lipoprotein, LDL (-), is an endogenously modified LDL subfraction with cytotoxic and proinflammatory actions on endothelial cells, monocytes, and macrophages contributing to the progression of atherosclerosis. In this study, epitopes of LDL (-) were mapped using a phage display library of peptides and monoclonal antibodies reactive to this modified lipoprotein. Two different peptide libraries (X6 and CX8C for 6- and 8-amino acid-long peptides, respectively) were used in the mapping. Among all tested peptides, two circular peptides, P1A3 and P2C7, were selected based on their high affinities for the monoclonal antibodies. Small-angle X-ray scattering analysis confirmed their structures as circular rings. P1A3 or P2C7 were quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFα, IL-1 ß and iNOS as well as the secretion of TNFα, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. In contrast, P1A3 did not show pro-inflammatory effects. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses.
Assuntos
Epitopos/metabolismo , Inflamação/metabolismo , Lipoproteínas LDL/metabolismo , Epitopos/sangue , Epitopos/isolamento & purificação , Humanos , Lipoproteínas LDL/sangue , Lipoproteínas LDL/isolamento & purificação , Macrófagos/metabolismo , Óxido Nítrico/análiseRESUMO
Porcine circovirus 2 (PCV2) is associated with a series of swine diseases. There is a great interest in improving our understanding of the immunology of PCV2, especially the properties of the viral capsid protein Cap-PCV2 and how they relate to the immunogenicity of the virus and the subsequent development of vaccines. Phage display screening has been widely used to study binding affinities for target proteins. The aim of this study was to use phage display screening to identify antigenic peptides in the PCV2 capsid protein. After the selection of peptides, five of them presented similarity to sequences found in cap-PCV2, and four peptides were synthesized and used for immunization in mice: 51-CTFGYTIKRTVT-62 (PS14), 127-CDNFVTKATALTY-138 (PS34), 164-CKPVLDSTIDY-173 (PC12), and 79-CFLPPGGGSNT-88 (PF1). Inoculation with the PC12 peptide led to the highest production of antibodies. Furthermore, we used the PC12 peptide as an antigen to examine the humoral response of swine serum by ELISA. The sensitivity and specificity of this assay was 88.9% and 92.85%, respectively. Altogether, characterization of immunogenic epitopes in the capsid protein of PCV2 may contribute to the improvement of vaccines and diagnostics.
Assuntos
Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Técnicas de Visualização da Superfície Celular , Circovirus/imunologia , Peptídeos/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/química , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/química , Circovirus/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Epitopos/isolamento & purificação , Camundongos , Testes de Neutralização , Peptídeos/química , Peptídeos/isolamento & purificação , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/sangue , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vacinas Virais/imunologiaRESUMO
Dengue is a global public health problem and is caused by four dengue virus (DENV) serotypes (DENV1-4). A major challenge in dengue vaccine development is that cross-reactive anti-DENV Abs can be protective or potentially increase disease via Ab-dependent enhancement. DENV nonstructural protein 1 (NS1) has long been considered a vaccine candidate as it avoids Ab-dependent enhancement. In this study, we evaluated survival to challenge in a lethal DENV vascular leak model in mice immunized with NS1 combined with aluminum and magnesium hydroxide, monophosphoryl lipid A + AddaVax, or Sigma adjuvant system+CpG DNA, compared with mice infected with a sublethal dose of DENV2 and mice immunized with OVA (negative control). We characterized Ab responses to DENV1, 2, and 3 NS1 using an Ag microarray tiled with 20-mer peptides overlapping by 15 aa and identified five regions of DENV NS1 with significant levels of Ab reactivity in the NS1 + monophosphoryl lipid A + AddaVax group. Additionally, we profiled the Ab responses to NS1 of humans naturally infected with DENV2 or DENV3 in serum samples from Nicaragua collected at acute, convalescent, and 12-mo timepoints. One region in the wing domain of NS1 was immunodominant in both mouse vaccination and human infection studies, and two regions were identified only in NS1-immunized mice; thus, vaccination can generate Abs to regions that are not targeted in natural infection and could provide additional protection against lethal DENV infection. Overall, we identified a small number of immunodominant regions, which were in functionally important locations on the DENV NS1 protein and are potential correlates of protection.
Assuntos
Antígenos Virais/imunologia , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Epitopos/imunologia , Proteínas não Estruturais Virais/imunologia , Adjuvantes Imunológicos , Adolescente , Animais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Reações Cruzadas , Dengue/epidemiologia , Dengue/virologia , Vírus da Dengue/química , Modelos Animais de Doenças , Epitopos/química , Epitopos/genética , Epitopos/isolamento & purificação , Feminino , Humanos , Imunidade Inata , Epitopos Imunodominantes/genética , Lactente , Masculino , Camundongos , Nicarágua/epidemiologia , Estudos Prospectivos , Sorotipagem , Vacinação , Proteínas não Estruturais Virais/químicaRESUMO
Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n = 31) compared to those from vaccinated dogs (n = 21), experimentally infected dogs with cross-reactive parasites (n = 23), and healthy controls (n = 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no cross-reactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programs.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários , Doenças do Cão/diagnóstico , Leishmania infantum/imunologia , Leishmaniose Visceral/veterinária , Biblioteca de Peptídeos , Peptídeos , Animais , Antígenos de Protozoários/isolamento & purificação , Brasil , Doenças do Cão/parasitologia , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/isolamento & purificação , Feminino , Leishmaniose Visceral/diagnóstico , Leishmaniose Visceral/parasitologia , Masculino , Peptídeos/isolamento & purificação , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Testes Sorológicos/métodosRESUMO
Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction in E. coli showed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera.
Assuntos
Epitopos , Hepatite B/diagnóstico , Proteínas Recombinantes , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/isolamento & purificação , Hepatite B/imunologia , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Temperatura Alta , Humanos , Imunoensaio , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Desdobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Fatores de TempoRESUMO
A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutination (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.
Assuntos
Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Camelídeos Americanos/imunologia , Epitopos/imunologia , Flagelina/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Lipoproteínas/imunologia , Animais , Antígenos de Bactérias/isolamento & purificação , Argentina/epidemiologia , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Camelídeos Americanos/sangue , Bovinos , Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Flagelina/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/imunologia , Lipoproteínas/isolamento & purificação , Testes Sorológicos/veterinária , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Se estudió un lote de 28 sueros de llama (Lama gama) de la provincia de Jujuy, Argentina, a fin de identificar antígenos inmunorreactivos contra Leptospira interrogans. Se utilizaron distintas preparaciones antigénicas de la bacteria para estudiar la inmunorreactividad mediante microaglutinación (MAT), ELISA y Western inmunoblot. Un pool de sueros bovinos positivos a la MAT fue empleado como control. Todos los sueros de llama fueron negativos mediante MAT e igual resultado se observó mediante ELISA. Dos de los 28 sueros de llama y el pool de sueros bovinos positivos, al ser evaluados por Western inmunoblot, arrojaron resultados positivos y permitieron identificar proteínas inmunorreactivas. Por MALDI-TOF se logró establecer que la proteína asociada a los dos sueros de llama inmunorreactivos era una flagelina periplásmica de Leptospira interrogans serovar Lai STR, mientras que la asociada al pool de sueros bovinos positivos a Leptospira sp. se trataba de una lipoproteína de la membrana externa de Leptospira interrogans serovar Ballum, LipL21. Estas proteínas podrían ser utilizadas en el diseño de un nuevo ELISA aplicado al diagnóstico temprano de leptospirosis, ya sea en distintos tipos de ganado como así también en reservorios silvestres.
A batch of 28 llama (Lama gama) sera from Jujuy province in Argentina was studied in order to identify immune reactive antigens to Leptospira interrogans. Different antigenic preparations from the bacterium were used to study the immune reactivity by the microagglutinattion (MAT), ELISA and Western immunoblot tests. A control pool of positive bovine sera was used. All the llama sera were negative to MAT as well as to ELISA. Two of the llama sera and the positive bovine sera pool rendered positive results when evaluated by Western immunoblot, allowing the identification of immune reactive proteins. These proteins were identified by MALDI-TOF. A periplasmic flagellin of Leptospira interrogans serovar Lai STR called FlaB1 was identified from the reactive llama sera, and an external membrane lipoprotein of Leptospira interrogans serovar Ballum called LipL21 was identified from the pool of bovine positive sera. These proteins could be used in a new ELISA applied to the early diagnosis of leptospirosis in different kind of cattle or wild reservoirs.
Assuntos
Animais , Bovinos , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Camelídeos Americanos/imunologia , Epitopos/imunologia , Flagelina/imunologia , Leptospira interrogans/imunologia , Leptospirose/veterinária , Lipoproteínas/imunologia , Antígenos de Bactérias/isolamento & purificação , Argentina/epidemiologia , Western Blotting , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Camelídeos Americanos/sangue , Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Flagelina/isolamento & purificação , Leptospirose/epidemiologia , Leptospirose/imunologia , Lipoproteínas/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Testes Sorológicos/veterináriaRESUMO
This work reports for the first time the identification and immunolocalization, by confocal and conventional indirect immunofluorescence, of m3G epitopes present in ribonucleoproteins of the following trypanosomatids: Trypanosoma cruzi epimastigotes of three different strains, Blastocrithidia ssp., and Leishmania major promastigotes. The identity of these epitopes and hence the specificity of the anti-m3G monoclonal antibody were ascertained through competition reaction with 7-methylguanosine that blocks the Ig binding sites, abolishing the fluorescence in all the parasites tested and showing a specific perinuclear localization of the snRNPs, which suggests their nuclear reimport in the parasites. Using an immunoprecipitation technique, it was also possible to confirm the presence of the trimethylguanosine epitopes in trypanosomatids.
Assuntos
Anticorpos Monoclonais , Epitopos/isolamento & purificação , Ribonucleoproteínas Nucleares Pequenas/isolamento & purificação , Trypanosomatina/química , Animais , Anticorpos Monoclonais/imunologia , Técnica Indireta de Fluorescência para Anticorpo , Imunoprecipitação , Microscopia Confocal , Ribonucleoproteínas Nucleares Pequenas/imunologia , Trypanosomatina/genética , Trypanosomatina/imunologia , Trypanosomatina/ultraestruturaRESUMO
Trichomonas vaginalis is a flagellated protozoan which infects the urogenital tract of humans. Previous studies have demonstrated that monoclonal antibodies (MAbs) against a 62 kDa proteinase (4D8 and 1A8) decreased cytoadherence of the parasite to epithelial cells in vitro and passive inoculation of mice with two MAbs 24 h before the intraperitoneal challenge resulted in different grade of protections to T. vaginalis infection. In the present paper we describe the characterization of the epitopes recognized by MAbs 4D8 and 1A8. The epitopes were characterized by heat treatment, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, enzymes proteolysis and periodate oxidation. The results showed that the two MAbs 4D8 and 1A8 each react with a different protein epitope of repetitive nature found on the same excretory-secretory molecules of T. vaginalis and it could explain the variation in the protection grade obtained in the challenge experiments.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Epitopos/imunologia , Vaginite por Trichomonas/prevenção & controle , Trichomonas vaginalis/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/isolamento & purificação , Antígenos de Protozoários/química , Antígenos de Protozoários/isolamento & purificação , Western Blotting , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/isolamento & purificação , Feminino , Camundongos , Peptídeo Hidrolases/imunologia , Peptídeo Hidrolases/isolamento & purificação , Vaginite por Trichomonas/imunologia , Trichomonas vaginalis/enzimologiaRESUMO
Several recombinant clones expressing antigens from Echinococcus granulosus were isolated previously from a parasite cDNA library using cystic hydatid disease (CHD) patients' sera or rabbit hyperimmune antiserum against a lipoproteic fraction from bovine cyst fluid. Six of these antigens were expressed in Escherichia coli and the purified recombinant proteins were tested in enzyme-linked immunosorbent assay (ELISA) for specific IgG with a panel of sera from patients with surgically confirmed (n = 58) or immunologically diagnosed (n = 71) CHD. Sera from clinically normal individuals (n = 203) and sera from individuals with other helminthic infections (n = 65) were assayed for the assessment of specificity. A cut-off value was determined by receiver-operating-characteristic plots for each antigen. A recombinant antigen B subunit (AgB8/2) presented the highest sensitivity (93.1%), considering the group of sera from patients with CHD surgically confirmed, and specificity (99.5%) and is proposed as the basis for an immunodiagnostic test. The other recombinant antigens tested presented sensitivities between 58.6% and 89.7%, and three of them were considered of complementary value. In subclass-specific ELISA, different IgG isotypes showed dominance in the response for each of the recombinant antigens. There was a clear predominance of IgG4 response for all antigens tested, indicating that this would be the subclass of choice to be assessed for these recombinant proteins.
Assuntos
Antígenos de Helmintos/isolamento & purificação , Equinococose/imunologia , Echinococcus/imunologia , Animais , Antígenos de Helmintos/imunologia , Área Sob a Curva , Estudos de Casos e Controles , Equinococose/diagnóstico , Epitopos/imunologia , Epitopos/isolamento & purificação , Escherichia coli/imunologia , Humanos , Imunoglobulina G/análise , Curva ROC , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Testes SorológicosRESUMO
This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Helmintos/química , Fasciola hepatica/imunologia , Animais , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/isolamento & purificação , Boroidretos/farmacologia , Bovinos , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Epitopos/imunologia , Epitopos/isolamento & purificação , Temperatura Alta , Imunoensaio , Lectinas/metabolismo , Mercaptoetanol/farmacologia , Neuraminidase/metabolismo , Ácido Periódico/farmacologia , Pronase/metabolismo , Ácido Tricloroacético/farmacologia , beta-Galactosidase/metabolismoRESUMO
We studied tha presence of antineutrophil cytoplasmic antibodies in 16 patients with idiopathic ulcerative colitis, using an indirect immunofluorescence technique and specific ELISA for myeloperoxidase and proteinase 3. Twelve patients had an active disease and in ten, antineutrophil cytoplasmic antibodies were positive, with a predominantly perinuclear distribution and without specificity for myeloperoxidase or proteinase 3. These antiantineutrophil cytoplasmic antibodies could be serologic indicators of disease activity in patients with ulcerative colitis
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Colite Ulcerativa/imunologia , Neutrófilos , Autoanticorpos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Anticorpos Antinucleares/isolamento & purificação , Epitopos/isolamento & purificação , ImunofluorescênciaRESUMO
The prevalence of antibodies against the repeat epitope of the circumsporozoite protein (cs) of the standard (PV210) and variant (PVK247) strain of Plasmodium vivax was determined by ELISA in 1170 sera from individual residents of seven localities of the Region Huasteca of San Luis Potosi, Mexico. The capture antigens were the synthetic peptides DDAAD and (ANGAGNQPG)4 that correspond to the repeats of the PV210 and PVK247 cs proteins, respectively. Of the analyzed serum samples, 34.1 percent (400/1170) were positive with one or both of these antigens. Of the sera, 18.2 percent (214/1170) reacted with the DDAADF peptide and 6.6 percent (78/1170) were positive with the variant synthetic peptide. Additionally, 9.2 percent (108/1170) of the samples reacted with both peptides. A sample of 10 percent of positive sera for the variant cs repeat (18/78) was tested with the cs repeat peptide of P. malariae/P. brasilianum (NAAG); almost all of them (16/18, 89 percent) being positive. These results confirm that the transmission of the variant strain of P. vivax is a common Phenomenon in endemic regions in Latin America, as well as in other tropical regions of the world. These findings may have implications for the development of a P. vivax vaccine since that based on the standard cs repeat only would not be universally protective
Assuntos
Ensaio de Imunoadsorção Enzimática , Epitopos/isolamento & purificação , Vacinas Antimaláricas/imunologia , México , Plasmodium vivax/imunologia , Proteínas de Protozoários/imunologiaRESUMO
A novel 58 kDa antigenic determinant of the fungus Paracoccidioides brasiliensis was identified by enzyme-linked immunosorbent assay using a panel of species-specific murine monoclonal antibodies (MAbs). Western immunoblot analysis, deglycosylation studies and isoelectric focusing indicated that this 58 kDa antigen is a glycoprotein, with a pI of approximately 5.2. The molecule was purified from P. brasiliensis culture filtrate and yeast cytoplasmic antigens by membrane ultrafiltration, liquid isoelectric focusing and gel filtration; N-terminal amino acid sequence data revealed no substantial homology with known proteins. The presence of the antigen in the cytoplasm of both yeast and mycelial forms of the fungus was demonstrated when these MAbs were used as markers in immunofluorescence, immunoperoxidase and immunoalkaline phosphatase techniques to label P. brasiliensis in cryostat sections. These MAbs also recognized the cytoplasm of P. brasiliensis yeast forms in paraffin-embedded pathological specimens from human cases. A preparation of the 58 kDa component from yeast cytoplasmic antigen was reacted by Western immunoblotting with 26 different serum samples from paracoccidioidomycosis patients, and 81% of them recognized it.
Assuntos
Antígenos de Fungos/análise , Epitopos/isolamento & purificação , Glicoproteínas/imunologia , Paracoccidioidomicose/imunologia , Adulto , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Western Blotting , Cromatografia em Gel , Ensaio de Imunoadsorção Enzimática , Humanos , Focalização Isoelétrica , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peso Molecular , Paracoccidioides/imunologiaRESUMO
Se estudiaron los niveles de anticuerpos contra epítopes Gal Ó 1,3 Gal en 407 sueros humanos chagásicos (92) y no chagásicos (315), mediante la reacción de hemaglutinación con eritrocitos de conejo; con inmunoelectrotransferencia se investigó la reactividad de sueros con altos títulos de anticuerpos anti-Gal frente a antígenos de escherichia coli y serratia marcescens. Finalmente, utilizando un anticuerpo anti-Gal purificado se identificó epítopes Gal Ó 1,3 Gal en formas metacíclicas de 12 cepas altoandinas chilenas de trypanosoma cruzi. Entre los 92 sueros chagásicos, se demostró que en el 68,5 por ciento (63) de los menos chagásicos se detectó anticuerpos anti-Gal a títulos ò 1:1.600, mientras que entre los sueros no chagásicos, sólo el 15,6 por ciento (49) mostró respuesta anti-Gal a títulos similares. Estos datos sugieren que la determinación de estos anticuerpos podría contribuir a complementar el diagnóstico de la infección, especialmente cuando se establezcan títulos de corte ò 1:3.200. La inmunoelectrotransferencia mostró que sueros de personas infectadas con T. cruzi reconocen varios antígenos presentes en E. coli y S. marcescens, lo que refuerza la idea de que a lo menos en parte estas bacterias serían capaces de estimular estas respuestas. El análisis autorradiográfico utilizando anticuerpo anti-Gal purificado, mostró diferencias en la expresión de los epítopes Gal Ó 1,3 Gal en las diferentes cepas de T. cruzi. Estos resultados sugieren que los anticuerpos anti-Gal podrían tener real significado en los mecanismos de inmunidad natural y protección de la infección en chilenos infectados con T. cruzi
Assuntos
Humanos , Masculino , Feminino , Gravidez , Recém-Nascido , Doença de Chagas/imunologia , Imunidade Inata , Soro Antilinfocitário/análise , Trypanosoma cruzi/imunologia , Doença de Chagas/sangue , Doença de Chagas/diagnóstico , Epitopos/isolamento & purificação , Escherichia coli/imunologia , Imunofluorescência , Imunossupressores , Serratia marcescens/imunologia , Soro Antilinfocitário/imunologia , Testes de Hemaglutinação , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
Protective immunity against rotavirus infection is directed against antigenic epitopes on the outer capsid proteins VP7 and VP4. The aim of this study was to characterize the VP7 and VP4 antigenic types circulating in different hospital areas of Santiago, Chile, obtained from children consulting for acute no bloody diarrhea in 5 hospitals representative of the 5 major health areas in Santiago. In addition, 256 rotavirus positive samples, obtained from children with acute diarrhea consulting in the north health area of Santiago between 1985-1987 were studied. All samples were processed for rotavirus by an ELISA and all rotavirus positive samples were selected for VP4 typing by PCR (types P1-P4). A total of 782 rotavirus positive samples were obtained of wich 618 (79 percent) were typeable for one specific VP7 type. VP7 type G1 represented 63 percent of the rotavirus positive samples and predominated in all areas evaluated throughout the entire period of observation. VP7 type G2 represented 13 percent of rotavirus samples, following G1 in predominance. G2 types decreased progressively in all areas in both study periods. G4 types were detected mainly during 1985-1987, and G3 types have so far not been detected. Preliminary analysis of VP4 types suggests that P1 types are predominant and closely associated with VP7 G1 type. These results are relevant for the adoption of appropiate preventive strategies for rotavirus infection, specifically aimed to the development of effective vaccines
Assuntos
Rotavirus/genética , Diarreia Infantil/microbiologia , Técnicas In Vitro , Manejo de Espécimes , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da Polimerase , Estudos Prospectivos , Estudos Retrospectivos , Epitopos/isolamento & purificaçãoRESUMO
Cation exchange chromatography was evaluated to purify the M antigen from histoplasmin (HMIN). Two H and M antigen-containing fractions, soluble (S) and precipitate (PP), resulted from the initial 0.025 M, pH 3.5 citrate buffer dialysis step. The PP fraction contained 62% of the M antigen activity and was resolubilized. Both fractions were chromatographed on CM Sepharose CL-6B. Polysaccharide C antigen was abundant in the S fraction and most of it did not bind to CM Sepharose. M antigen-enriched fractions were eluted with 0.5 M NaCl. Re-chromatography of the relevant S fraction (S-II) and PP fraction (PP-II) by linear gradient fast protein liquid chromatography (FPLC) removed protein and C impurities. M antigen purified by FPLC from the PP-II fraction was depleted of other antigens when Western blots were probed with anti-M, anti-H and anti-C monoclonal antibodies (Mabs). M antigen was identified as a 94 kDa glycoprotein containing a specific-protein epitope and an epitope that reacted with a Mab against the polysaccharide C antigen. M antigen can be purified from HMIN by tandem cation exchange chromatography of the precipitable fraction on an open CM Sepharose CL-6B column followed by linear gradient FPLC.