RESUMEN
Hepatitis B virus (HBV) infection is the major public health problem leading cause of death worldwide. The most important diagnostic marker for this infection is hepatitis B surface antigen (HBsAg). In this study, a novel, inexpensive, portable and sensitive ELISA method was designed and investigated for diagnosis of HBsAg based on the functionalized Fe3O4 and Al2O3 nanoparticles, with the strategy for detecting the concentration of glucose using a cheap and accessible personal glucose meter (PGM). The ELISA system was constructed using hepatitis B antibody against HBsAg immobilized on streptavidin coated magnetic iron oxide particles (S-Fe3O4) as the capture antibody (Ab1). In addition, another hepatitis B antibody against different epitope of HBsAg (Ab2) and glucoamylase both were immobilized on Al2O3 nanoparticles. After formation of the sandwich immune complex between Ab1 and Ab2 immobilized on S-Fe3O4 and Al2O3 NPs, respectively, through HBsAg, starch was converted into glucose using glucoamylase. Then, the glucose concentration was measured using PGM. The concentration of HBsAg was calculated based on the linear relation between the concentrations of HBsAg and glucose. Under optimal conditions, this assay showed detection limit values of 0.3 to 0.4â¯ngâ¯ml-1 for "ay" and "ad" subtypes of HBsAg, respectively. The results indicate that the designed assay is comparable to the commercial kits in terms of sensitivity, on-site, specificity, cost, simplicity, portability and reproducibility. The presented method can be used in disadvantaged areas of the world and blood transfusion centers. To the best of our knowledge, this is the first report of using PGMs for HBSAg detection.
Asunto(s)
Técnicas Biosensibles/instrumentación , Glucemia/análisis , Antígenos de Superficie de la Hepatitis B/aislamiento & purificación , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/diagnóstico , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Técnicas Biosensibles/economía , Técnicas Biosensibles/métodos , Glucemia/metabolismo , Ensayo de Inmunoadsorción Enzimática/economía , Ensayo de Inmunoadsorción Enzimática/instrumentación , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/inmunología , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Hepatitis B/sangre , Hepatitis B/virología , Anticuerpos contra la Hepatitis B/química , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Humanos , Límite de Detección , Nanopartículas de Magnetita/química , Juego de Reactivos para Diagnóstico/economía , Reproducibilidad de los ResultadosRESUMEN
Substitution of the defective lysosomal enzyme in lysosomal storage disorders (LSDs) often elicits antibody formation towards the infused protein. Aside from Gaucher disease, antibodies often lead to infusion associated reactions and a reduced biochemical response. In Pompe disease, antibody titer is predictive of clinical outcome, but this is less apparent in other LSDs and warrants further study. Few laboratories are capable of enzyme-antibody determination: often physicians need to rely on the enzyme manufacturer for analysis. Currently, laboratories employ different antibody assays which hamper comparisons between cohorts or treatment regimens. Assay standardisation, including measurement of antibody-related enzyme inhibition, is therefore urgently needed. Successful immunomodulation has been reported in Pompe and in Gaucher disease, with variable success. Immunomodulation regimens that contain temporary depletion of B-cells (anti-CD20) are most used. Bone marrow transplantation in MPS-I results in disappearance of antibodies. No other clinical studies have been conducted in humans with immunomodulation in other LSDs.
Asunto(s)
Terapia de Reemplazo Enzimático , Terapia Enzimática , Isoanticuerpos/inmunología , Enfermedades por Almacenamiento Lisosomal/tratamiento farmacológico , Anticuerpos/inmunología , Enzimas/inmunología , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/inmunología , Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/inmunología , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/uso terapéutico , Glucosilceramidasa/inmunología , Glucosilceramidasa/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Enfermedad del Almacenamiento de Glucógeno Tipo II/inmunología , Humanos , Enfermedades por Almacenamiento Lisosomal/inmunología , alfa-Galactosidasa/inmunología , alfa-Galactosidasa/uso terapéuticoRESUMEN
Antibodies formed against the therapeutic protein are a life-threatening complication that arises during enzyme replacement therapy for Pompe disease (acid α-glucosidase deficiency; GAA). To provide an effective alternative to current practices, we investigated the capacity of anti-B-cell activating factor (BAFF) as a novel drug candidate to prevent antibody formation in a Pompe disease mouse model. A BAFF-neutralizing antibody was administered prophylactically and with maintenance doses in association with enzyme replacement therapy using recombinant human GAA in Gaa(-/-) mice. BAFF blockade delayed antibody production and increased GAA activity within tissues with protection from anaphylaxis. Anti-BAFF also resolved antibody formation during an immune response and precluded the maturation of antibody secreting cells from entering the bone marrow compartment. This treatment modality may therefore be a viable alternative for the clinical management of antibody formation for Pompe disease and has potential use against antibody formation in other protein replacement therapies.
Asunto(s)
Anticuerpos/inmunología , Factor Activador de Células B/antagonistas & inhibidores , Terapia de Reemplazo Enzimático , Glucano 1,4-alfa-Glucosidasa/uso terapéutico , Enfermedad del Almacenamiento de Glucógeno Tipo II/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Terapia Genética/métodos , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Masculino , Ratones , Ratones Noqueados , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
BACKGROUND: Schizophyllum commune is one of the causative agents of basidiomycosis including disorders such as allergic bronchopulmonary mycosis, allergic fungal sinusitis, and mucoid impaction of bronchi, the incidence of those of which has been increasing. These mycoses are difficult to diagnose because only a limited number of diagnostic tools are currently available. The biggest problem is that no specific antigens of S. commune have been identified to enable serodiagnosis of the disease. OBJECTIVE: In this study, we attempted to identify a major antigen of S. commune to establish a reliable serodiagnostic method. METHODS: We used mass spectrometry to identify an antigen that reacted with the serum of a patient with allergic bronchopulmonary mycosis caused by S. commune. The protein was expressed in Escherichia coli, highly purified, and the patient sera IgG and IgE titres against the protein were determined by enzyme-linked immunosorbent assay. RESULTS: The protein identified as a major antigen of S. commune was named Sch c 1; it was a homolog of glucoamylase. The IgG and IgE titres against Sch c 1 in patient sera were significantly higher than those in healthy volunteer sera (P < 0.01). CONCLUSIONS AND CLINICAL RELEVANCE: Sch c 1 is recognized by the host immune system of patients as an antigen/allergen. The purified glucoamylase Sch c 1 is a promising candidate antigen for the serodiagnosis of S. commune-induced mycosis.
Asunto(s)
Alérgenos/inmunología , Antígenos Fúngicos/inmunología , Glucano 1,4-alfa-Glucosidasa/inmunología , Micosis/inmunología , Schizophyllum/inmunología , Alérgenos/química , Secuencia de Aminoácidos , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/inmunología , Antígenos Fúngicos/química , Reacciones Cruzadas/inmunología , Glucano 1,4-alfa-Glucosidasa/química , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Datos de Secuencia Molecular , Micosis/sangre , Aspergilosis Pulmonar/inmunología , Schizophyllum/enzimología , Alineación de SecuenciaAsunto(s)
Alérgenos/análisis , Contaminación de Medicamentos , Glucano 1,4-alfa-Glucosidasa/inmunología , Neurospora crassa/enzimología , Neurospora crassa/inmunología , Alérgenos/inmunología , Secuencia de Aminoácidos , Humanos , Inmunoglobulina E/sangre , Datos de Secuencia Molecular , Neurospora crassa/genética , VacunasRESUMEN
Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA(-/-) mice receiving 1 x 10(12) vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.
Asunto(s)
Dependovirus/genética , Terapia Genética , Glucano 1,4-alfa-Glucosidasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/inmunología , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Animales , Anticuerpos Monoclonales/sangre , Femenino , Vectores Genéticos/administración & dosificación , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/fisiología , Glucógeno/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Humanos , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Miocardio/enzimología , Miocardio/patología , alfa-GlucosidasasRESUMEN
Autosomal recessive deficiency of lysosomal acid maltase (GAA) or glycogen storage disease type II (GSDII) results in a spectrum of phenotypes including a rapidly fatal infantile disorder (Pompe's), juvenile, and a late-onset adult myopathy. The infantile onset form presents as hypotonia with massive accumulation of glycogen in skeletal and heart muscle, with death due to cardiorespiratory failure. Adult patients with the slowly progressive form develop severe skeletal muscle weakness and respiratory failure. Particle bombardment is a safe, efficient physical method in which high-density, subcellular-sized particles are accelerated to high velocity to carry DNA into cells. Because it does not depend on a specific ligand, receptor, or biochemical features on cell surfaces, particle-mediated gene transfer can be readily applied to a variety of systems. We evaluated particle bombardment as a delivery system for therapy of GSDII. We utilized a vector carrying the CMV promoter linked to the human GAA cDNA. Human GSDII cell lines (fibroblasts and lymphoid) as well as ex vivo with adult-onset peripheral blood cells (lymphocytes and monocytes) were transiently transfected by bombardment with a Helios gene gun delivering gold particles coated with the GAA expression plasmid. All cell types showed an increase in human GAA activity greater than 50% of normal activity. Subsequently, GAA -/- mice were treated every 2 weeks for 4 months by particle bombardment to the epidermis of the lower back and hind limbs. Muscle weakness in the hind and forelimbs was reversed. These data suggest that particle delivery of the GAA cDNA by the Helios gene gun may be a safe, effective treatment for GSDII.
Asunto(s)
Biolística , Glucano 1,4-alfa-Glucosidasa/genética , Enfermedad del Almacenamiento de Glucógeno Tipo II/terapia , Adulto , Animales , Formación de Anticuerpos , Línea Celular , Citomegalovirus/genética , ADN Complementario/administración & dosificación , ADN Complementario/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Glucano 1,4-alfa-Glucosidasa/sangre , Glucano 1,4-alfa-Glucosidasa/inmunología , Enfermedad del Almacenamiento de Glucógeno Tipo II/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Humanos , Técnicas In Vitro , Recién Nacido , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , alfa-GlucosidasasRESUMEN
A 125-kDa starch hydrolysing enzyme of Aspergillus niger characterised by its ability to dextrinise and saccharify starch [Suresh et al. (1999) Appl. Microbiol. Biotechnol. 51, 673-675] was also found to possess activity towards raw starch. Segregation of these activities in the 71-kDa glucoamylase and a 53-kDa alpha-amylase-like enzyme supported by antibody cross-reactivity studies and the isolation of mutants based on assay screens for the secretion of particular enzyme forms revealed the 125-kDa starch hydrolysing enzyme as their precursor. N-terminal sequence analysis further revealed that the 71-kDa glucoamylase was the N-terminal product of the precursor enzyme. Immunological cross reactivity of the 53-kDa amylase with antibodies raised against the precursor enzyme but not with the 71- and 61-kDa glucoamylase antibodies suggested that this enzyme activity is represented by the C-terminal fragment of the precursor. The N-terminal sequence of the 53-kDa protein showed similarity to the reported Taka amylase of Aspergillus oryzae. Antibody cross-reactivity to a 10-kDa non-enzymic peptide and a 61-kDa glucoamylase described these proteins as products of the 71-kDa glucoamylase. Identification of only the precursor starch hydrolysing enzyme in the protein extracts of fungal protoplasts suggested proteolytic processing in the cellular periplasmic space as the cause for the secretion of multiple forms of amylases by A. niger.
Asunto(s)
Aspergillus niger/enzimología , Aspergillus niger/metabolismo , Precursores Enzimáticos/metabolismo , Glucano 1,4-alfa-Glucosidasa/metabolismo , Procesamiento Proteico-Postraduccional , alfa-Amilasas/metabolismo , Secuencia de Aminoácidos , Anticuerpos/inmunología , Aspergillus niger/citología , Aspergillus niger/genética , Aspergillus oryzae/enzimología , Western Blotting , Reacciones Cruzadas/inmunología , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/inmunología , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/inmunología , Focalización Isoeléctrica , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Periplasma/enzimología , Periplasma/metabolismo , Protoplastos/citología , Protoplastos/enzimología , Protoplastos/metabolismo , Almidón/metabolismo , Almidón/ultraestructura , alfa-Amilasas/química , alfa-Amilasas/genética , alfa-Amilasas/inmunologíaRESUMEN
Functionally important carboxyl groups in the glucoamylase, Gluc1, from Rhizopus niveus were investigated by site-specific modification using water-soluble carbodiimide, 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide, and nucleophiles. A total of 7.5 carboxyl groups of the 37 present in Gluc1 were substituted in the presence of the substrate maltose, and there was a slight loss of enzymatic activity. After removal of maltose, re-treatment of the deprotected enzyme reduced its activity to 3% with the modification of 1.2 carboxyl groups. It is conceivable that there is only one carboxyl group located in the active site. Fluorescence spectra of the enzyme suggested an interaction of tryptophan residues and carboxyl groups at the locus of the enzyme active site. Distinctive disruption of the protein structure in the modified enzyme was excluded by CD spectroscopy and immunological investigation.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/química , Rhizopus/enzimología , Triptófano/química , Sitios de Unión , Carbodiimidas/química , Dicroismo Circular , Activación Enzimática , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/aislamiento & purificación , Glucano 1,4-alfa-Glucosidasa/metabolismo , Concentración de Iones de Hidrógeno , Inmunodifusión , Cinética , LigandosAsunto(s)
Aspergillus niger/enzimología , Cromatografía de Afinidad/métodos , Enzimas/metabolismo , Calor , Anticuerpos , Western Blotting , Estabilidad de Enzimas , Enzimas/inmunología , Enzimas/aislamiento & purificación , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/aislamiento & purificación , Glucano 1,4-alfa-Glucosidasa/metabolismo , alfa-Glucosidasas/inmunología , alfa-Glucosidasas/aislamiento & purificación , alfa-Glucosidasas/metabolismoRESUMEN
BACKGROUND: Aspergillus-derived enzymes are used in dough improvers in bakeries. Some of these enzymes are identified as causing IgE-mediated sensitization in up to 25% of bakers with workplace-related symptoms. OBJECTIVE: The aim of this study was to compare the frequency of sensitization to Aspergillus xylanase, cellulase, and glucoamylase with the sensitization to alpha-amylase (Asp o 2) and to identify IgE-reactive proteins in enzyme preparations. METHODS: Sensitization to Aspergillus-derived enzymes and cross-reactivity were retrospectively studied by enzyme allergosorbent test (EAST) and EAST-inhibition experiments. IgE-reactive proteins were detected by electrophoretic separation and immunoblotting. Liquid chromatography with electrospray ionization mass spectrometry and Edman degradation of tryptic protein fragments were used for the biochemical identification of an unknown IgE-binding protein. RESULTS: Twenty-three percent of 171 tested bakers had specific IgE to alpha-amylase, 8% reacted to glucoamylase, 13% reacted to cellulase, and 11% reacted to xylanase. Xylanase and cellulase preparations, each containing at least 6 different proteins, showed cross-reactivity in the range of 80%. The main IgE-binding protein in the xylanase preparation recognized in 7 of 8 xylanase-positive subjects was a protein of about 105 kd. This protein was identified as beta-xylosidase by peptide mass spectrometric fingerprinting. The identification was confirmed by matching 12 peptide sequences obtained by N-terminal and mass spectrometric sequencing to this protein. CONCLUSIONS: Beta-Xylosidase from Aspergillus niger is an occupational allergen present in currently used baking additives, which causes sensitization in at least 4% of symptomatic bakers. According to the International Union of Immunological Societies nomenclature, we suggest the term Asp n 14 for this allergen.
Asunto(s)
Alérgenos/inmunología , Antígenos Fúngicos , Aspergillus niger/inmunología , Manipulación de Alimentos , Hipersensibilidad/inmunología , Enfermedades Profesionales/inmunología , Xilosidasas/inmunología , Adulto , Alérgenos/clasificación , Secuencia de Aminoácidos , Antígenos de Plantas , Aspergillus niger/enzimología , Celulasa/inmunología , Reacciones Cruzadas , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Femenino , Glucano 1,4-alfa-Glucosidasa/inmunología , Humanos , Hipersensibilidad/sangre , Immunoblotting , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Enfermedades Profesionales/sangre , Estudios Retrospectivos , Dodecil Sulfato de Sodio , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/química , Xilosidasas/clasificación , alfa-Amilasas/inmunologíaRESUMEN
In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1-8 of mature glucoamylase greatly enhance excretion of both glucoamylase and beta-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro. Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/metabolismo , Péptidos/genética , Péptidos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismo , Transporte Biológico/genética , Western Blotting , Extractos Celulares/análisis , Clonación Molecular , Medios de Cultivo/análisis , Medios de Cultivo/metabolismo , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Glucano 1,4-alfa-Glucosidasa/inmunología , Mutagénesis Sitio-Dirigida , Plásmidos , Eliminación de Secuencia , Transducción de Señal/genética , beta-Galactosidasa/inmunologíaRESUMEN
Seven Arxula adeninivorans strains originating from The Netherlands (CSIR 1136, CSIR 1138 and CBS 8244T), South Africa (CSIR 1147, CSIR 1148 and CSIR 1149) and Siberia (Ls3) were compared concerning their secretory glucoamylase. Within the spectrum of secretory polypeptides obtained by SDS-polyacrylamide gel electrophoresis, the glucoamylase corresponding polypeptide, could be identified by means of specific antibodies directed against the glucoamylase of strain Ls3. The molecular masses of the glucoamylases vary from 84 to 95 kDa. After endoglycosidase F treatment of the secretory proteins, the N-linked carbohydrate content for each glucoamylase could be quantified at about 25%. Glucoamylase activity staining of secretory proteins separated under nondenaturing conditions revealed one glucoamylase active protein for the Dutch strains and two active forms for the strains originating from South Africa and Siberia. Highest activities of glucoamylase can be measured at the end of the exponential growth phase for each strain. The Dutch strain CSIR 1138 secretes the highest activity. Optimum values for temperature and pH were determined and compared. By means of pulsed field gel electrophoresis and Southern analysis, the glucoamylase corresponding gene could be localized on the second of the four chromosomes of each yeast strain.
Asunto(s)
Glucano 1,4-alfa-Glucosidasa/metabolismo , Hongos Mitospóricos/enzimología , Anticuerpos Antifúngicos/biosíntesis , Mapeo Cromosómico , Glucano 1,4-alfa-Glucosidasa/genética , Glucano 1,4-alfa-Glucosidasa/inmunología , Hongos Mitospóricos/genética , Hongos Mitospóricos/inmunologíaRESUMEN
The presence of the enzymatically active allergens equivalent to Der p I (cysteine protease), Der p III (serine protease) and amylase in extracts of Dermatophagoides pteronyssinus, D. farinae and Euroglyphus maynei was determined using appropriate enzymatic techniques. Biochemical equivalents of all three allergens were present in each extract studied. Studies also showed that the mite extracts contained a variety of other biochemically active enzymes including trypsin, chymotrypsin, carboxypeptidase A and B, glucoamylase and lysozyme. Marked differences in the relative concentrations of some of these enzymes in different mite extracts were observed, particularly trypsin and carboxypeptidase A. The enzymes were physicochemically similar to equivalent enzymes from vertebrate and invertebrate sources. Chromatofocusing studies of faecal extracts derived from D. pteronyssinus and D. farinae showed that several isoforms of each enzyme were present. The data indicated that there were more trypsin isoforms, with pI over a wider range, in extracts prepared from D. pteronyssinus. Proteases and carbohydrases were also found in extracts prepared from faecally enriched material suggesting that they were endoperitrophic and associated with mite digestion. The data suggest that not only are the group I, III and amylase allergens a consistent feature of most pyroglyphid dust mites but also that other proteases and carbohydrases present in mite faeces are allergenic.
Asunto(s)
Alérgenos/análisis , Endopeptidasas/inmunología , Glicósido Hidrolasas/inmunología , Ácaros/enzimología , Amilasas/química , Amilasas/inmunología , Amilasas/metabolismo , Animales , Endopeptidasas/química , Endopeptidasas/metabolismo , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/inmunología , Glucano 1,4-alfa-Glucosidasa/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/metabolismo , Ácaros/inmunología , Muramidasa/química , Muramidasa/inmunología , Muramidasa/metabolismo , Especificidad por Sustrato , Tripsina/química , Tripsina/inmunología , Tripsina/metabolismoAsunto(s)
Demencia/diagnóstico , Epilepsia/diagnóstico , Glucano 1,4-alfa-Glucosidasa/inmunología , Anciano , Análisis Químico de la Sangre , Encéfalo/patología , Comorbilidad , Demencia/complicaciones , Epilepsia/etiología , Epilepsia/fisiopatología , Salud de la Familia , Femenino , Glucano 1,4-alfa-Glucosidasa/metabolismo , Glucano 1,4-alfa-Glucosidasa/farmacocinética , Humanos , Hipotonía Muscular , Trastornos de la Personalidad/complicaciones , Trastornos de la Personalidad/psicología , Derivación y Consulta , Tomografía Computarizada por Rayos XRESUMEN
In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.
Asunto(s)
Anticuerpos , Estabilidad de Enzimas , Glucano 1,4-alfa-Glucosidasa/inmunología , alfa-Amilasas/inmunología , Animales , Complejo Antígeno-Anticuerpo , Glucano 1,4-alfa-Glucosidasa/metabolismo , Humanos , Cinética , Ratones/inmunología , Conejos/inmunología , Termodinámica , alfa-Amilasas/metabolismoRESUMEN
The effectiveness of a methodology designed to protect the antigen binding capacity of monoclonal antibodies undergoing labelling with a number of reagents was examined. The antigen binding sites of monoclonal antibodies were protected by complexing them with their antigen. Chemical modification with 6 mM of the water soluble Bolton-Hunter reagent of site protected monoclonal antibodies to glucoamylase resulted in antibodies that could tolerate a four-fold increase in reagent incorporation, without any loss of antigen binding capacity. Iodination of these antibodies (modified under site protected conditions) yielded over 70% increase in radioactivity incorporated in the active antibody fraction, compared with the incorporation into unprotected antibodies. Site protected labeling was found to be effective in retaining the antigen binding capacity of monoclonal antibodies modified with all reagents tested with the exception of chloramine-T.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/inmunología , Marcaje Isotópico/métodos , Animales , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Glucano 1,4-alfa-Glucosidasa/inmunología , Radioisótopos de Yodo , Ratones , SuccinimidasRESUMEN
A chemical synthesis of N-tris (beta-D-galactopyranosyloxymethyl) glycine methylamide (trisgalactosylglycine) has been carried out. Trisgalactosylglicine derivatives of bovine serum albumin, ovalbumin and amyloglucosidase from Aspergillus have been obtained. The binding of trisgalactosylglycine residues to bovine serum albumin and ovalbumin was performed by the carbodiimide method; amyloglucosidase galactosylation was performed by using the reductive amination method. The latter technique seems to be the most mild one because it does not interfere with the peptide structure of the protein being analyzed. The antiserum specifically raised against the trisgalactosylglycine derivative of bovine serum albumin as well as the monospecific antibodies isolated from it can interact with both the antigen and the trisgalactosylglycine derivatives of ovalbumin and amyloglucosidase. Native proteins are not precipitated with this antiserum. This suggests that the trigalactosylglycine residues (bovine serum albumin, ovalbumin, amyloglucosidase) covalently bound to various proteins act as immunologic determinants regardless of the mode of their binding.
Asunto(s)
Epítopos/química , Galactósidos/inmunología , Glicina/análogos & derivados , Glicoproteínas/inmunología , Animales , Cromatografía de Afinidad , Galactósidos/síntesis química , Galactósidos/química , Glucano 1,4-alfa-Glucosidasa/química , Glucano 1,4-alfa-Glucosidasa/inmunología , Glicina/síntesis química , Glicina/química , Glicina/inmunología , Inmunización , Espectroscopía de Resonancia Magnética , Ovalbúmina/química , Ovalbúmina/inmunología , Pruebas de Precipitina , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/inmunologíaRESUMEN
Use of antibodies towards neutral alpha-glucosidase from human kidney brush border enabled to estimate distinctly the activity of acid alpha-glucosidase in urine of healthy persons and patients although the activity of neutral enzyme in urine exceeded markedly the acid enzyme activity. Simultaneous use of antibodies to both these enzymes permitted to estimate separately the activity of acid and neutral alpha-glucosidases in a mixture. Accuracy of these estimations was confirmed after consecutive use of these antibodies.
Asunto(s)
Pruebas Enzimáticas Clínicas , Glucano 1,4-alfa-Glucosidasa/orina , Glucosidasas/orina , Sueros Inmunes , alfa-Glucosidasas/orina , Glomerulonefritis/diagnóstico , Glucano 1,4-alfa-Glucosidasa/inmunología , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Humanos , Pruebas de Precipitina , alfa-Glucosidasas/inmunologíaRESUMEN
In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.