RESUMEN
BACKGROUND: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. METHODS: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS-PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. RESULTS: In 1D analysis, 86% of sera showed binding to Fra e 1 (18-20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to beta-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. CONCLUSIONS: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization.
Asunto(s)
Fraxinus/inmunología , Inmunoglobulina E/sangre , Rinitis Alérgica Estacional/sangre , Rinitis Alérgica Estacional/inmunología , Western Blotting , Reacciones Cruzadas/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina E/inmunología , Proteómica , Rinitis Alérgica Estacional/epidemiología , Pruebas Cutáneas , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.
Asunto(s)
Alérgenos/inmunología , Carbohidratos/inmunología , Hipersensibilidad Inmediata/inmunología , Péptidos/inmunología , Extractos Vegetales/inmunología , Polen/inmunología , Alérgenos/química , Alérgenos/aislamiento & purificación , Arabidopsis/química , Western Blotting , Brassica napus/química , Carbohidratos/química , Carbohidratos/aislamiento & purificación , Reacciones Cruzadas/inmunología , Dactylis/química , Electroforesis en Gel Bidimensional , Epítopos/inmunología , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/química , Péptidos/química , Péptidos/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Polen/químicaRESUMEN
BACKGROUND: The Arabidopsis thaliana genome was recently fully sequenced, and this plant is now considered as the most useful model to study the effects of genetic engineering. The aim of the present study was to identify A. thaliana IgE-binding molecules and to localize their genes in order to evaluate the potential effect of gene insertion on the expression of IgE-binding molecules. METHODS: A. thaliana flower proteins were separated by two-dimensional gel electrophoresis and transferred onto a nitrocellulose sheet. The nitrocellulose sheet was successively incubated with human sera known to contain IgE that binds to rapeseed proteins, alkaline phosphatase-conjugated goat anti-human IgE and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. One allergen was further identified by N-terminal amino acid microsequencing. RESULTS: The results showed that some individuals possessed IgE that recognized numerous proteins with high molecular masses and various isoelectric points. This binding pattern strongly suggests that the epitopes recognized by these IgE could be, at least partly, sugar residues. Otherwise, out of the 10 sera that possessed IgE to Arabidopsis flower proteins, one serum strongly recognized a unique basic protein with an apparent molecular mass of around 14 kD. This protein was identified by amino acid microsequencing as the lipid transfer protein 1 (LTP1). CONCLUSION: We have demonstrated that A. Thaliana LTP1 is IgE reactive. The gene encoding this protein is located on chromosome 2, but it has been described that family 1 of A. Thaliana LTPs constitutes a multigenic family with genes located on various chromosomes.
Asunto(s)
Alérgenos/genética , Alérgenos/inmunología , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Secuencia de Aminoácidos , Antígenos de Plantas , Flores/inmunología , Galectina 3/genética , Galectina 3/inmunología , Expresión Génica , Genoma de Planta , Humanos , Hipersensibilidad/inmunología , Técnicas In Vitro , Datos de Secuencia Molecular , Proteínas de Plantas , Polen/inmunologíaRESUMEN
BACKGROUND: Type I hypersensitivity to rapeseed pollen allergens was described as the result of a cross-sensitization with various pollens that could constitute an aggravating factor in birch or grass pollen allergies. Recently, a few rapeseed pollen allergens were described. The aim of the present work was to identify new rapeseed pollen allergens by using two-dimensional gel analysis, microsequencing, and mass spectrometry. METHODS: Water extractable proteins from oilseed rape pollen or stamen were separated by two-dimensional gel electrophoresis. The proteins were then electroblotted onto a nitrocellulose (NC) sheet. The NC sheets were successively incubated with (1) individual human sera pre-selected for their immunoglobulin E (IgE) reactivity to rapeseed pollen proteins, (2) alkaline phosphatase (AP)-conjugated goat anti-human IgE and (3) AP substrate. The allergens localized by this method were then identified by microsequencing and MALDI-TOF mass spectrometry analysis. RESULTS: Of the 18 sera studied, five recognized a wide multispot zone with a molecular mass around 43 kD and pIs between 6.5 and 8.5. The results obtained with two representative sera are shown. From this zone, two isoforms of the polygalacturonase enzyme were identified by microsequencing. Confirmation was obtained through MALDI-TOF mass spectrometry analysis. CONCLUSION: The present results allow the identification of a new rapeseed allergen that can be the main allergen for some patients.
Asunto(s)
Alérgenos/inmunología , Brassica rapa/inmunología , Aceites de Plantas , Polen/inmunología , Poligalacturonasa/inmunología , Alérgenos/análisis , Western Blotting , Electroforesis en Gel Bidimensional , Ácidos Grasos Monoinsaturados , Humanos , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Isoenzimas/análisis , Espectrometría de Masas , Proteínas de Plantas/inmunología , Aceite de Brassica napusRESUMEN
BACKGROUND/OBJECTIVE: Latex allergy is a type 1 hypersensitivity reaction that mainly affects high-risk populations such as health care workers, spina bifida-affected or multiply-operated children. Ten molecules have so far been identified and registered as latex allergens (Hev b 1 to Hev b 10). The aim of the present investigation was to identify the major latex allergens by an individual analysis of the IgE response of latex-allergic patients to latex proteins separated by two-dimensional (2-D) gel electrophoresis. MATERIALS AND METHODS: Latex proteins from a sap or a glove extract were separated by 2-D electrophoresis and transferred to a nitrocellulose membrane. Each membrane was incubated with the serum of one latex-allergic patient. The most frequently recognized latex allergens were characterized in sap and glove extracts using monoclonal antibodies or amino acid microsequencing. RESULTS: The one-dimensional screening of 54 patient sera revealed 4 major bands recognized by IgE. The 2-D analysis of the sensitization to latex allergens allows the identification of allergen isoforms and the characterization of an individual response diversity. Hev b 6.01 was recognized by 88.9% of the patients. Protein spots around 14 kD were recognized by 48.1% of the patients and corresponded to Hev b 6.03 as well as other proteins. A not yet characterized doublet of acidic proteins with molecular masses of 43 and 94 kD was recognized by 20.4% of the sera. Only 5.5% of the sera did not recognize any of these 4 major allergens. Hev b 1 is the main protein from the glove extract but was not constantly found in sap extracts. CONCLUSIONS: One-dimensional electrophoretic analysis of the allergen is usually not sufficient to characterize the individual specificity of the IgE response to latex allergens. Latex-glove proteins which are allergens can be absent from the sap extracts and the sensitization to these allergens could be underestimated. Individual 2-D analysis of the sensitization to latex allergens is useful to define the best allergen mixture required for diagnosis and needed for individual therapy monitoring.
Asunto(s)
Alérgenos/química , Alérgenos/inmunología , Hipersensibilidad al Látex/inmunología , Látex/química , Látex/inmunología , Alérgenos/genética , Electroforesis en Gel Bidimensional , Guantes Protectores , Humanos , Inmunoglobulina E/sangre , Hipersensibilidad al Látex/terapia , Proteínas de Plantas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
BACKGROUND: Oilseed rape pollen allergies have been previously described as the result of cross-sensitization with various pollens. Recently, several proteins have been identified as oilseed rape allergens. The aim of the present work was the characterization of oilseed rape pollen allergens by two-dimensional (2-D) gel analysis and amino acid microsequencing. METHODS: Water extractable proteins from oilseed rape pollen were separated by isoelectrofocusing and then transferred onto a nitrocellulose sheet. Twenty-one human sera from pollen- or mustard-allergic individuals were screened for their reactivity to oilseed rape proteins. Eleven sera possessed IgE which recognized oilseed rape pollen proteins and one serum was selected for further 2-D characterization and amino acid microsequencing of the allergens. RESULTS: The results showed that three molecules from oilseed rape pollen were identified as oilseed rape allergens which have not yet been described. These three proteins were molecules of 70 kD with a pI >8, 40 kD with a pI around 10 and 80 kD with a pI around 5. These proteins displayed identities with the berberine bridge protein, a receptor-like protein kinase and the cobalamin-independent methionine synthetase from Arabidopsis thaliana, respectively. The genes encoding the putative Arabidopsis molecules are located on chromosome 1 (berberine bridge protein) and chromosomes 3 and 4 (receptor-like protein kinases). CONCLUSION: These results show that certain high-molecular-mass proteins from oilseed rape pollen are allergens.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/aislamiento & purificación , Alérgenos/aislamiento & purificación , Brassica/inmunología , Proteínas de Plantas/aislamiento & purificación , Polen/química , Proteínas Quinasas/aislamiento & purificación , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Western Blotting , Brassica/química , Brassica/genética , Electroforesis en Gel Bidimensional , Humanos , Inmunoglobulina E/inmunología , Focalización Isoeléctrica , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Polen/inmunología , Proteínas Quinasas/química , Proteínas Quinasas/genética , Prueba de Radioalergoadsorción , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Antisperm antibodies eluted from the surface of spermatozoa obtained from infertile men recognised several common epitopes. We tested whether these epitopes were relevant to fertility by isolating the immunodominant 37/36 and 19/18 protein zones. These protein zones were cut out of preparative slab gels and electro-eluted. The isolated proteins, P36 and P18, were used for biochemical characterisation and to produce specific antibodies in rabbits. The specific reactivity of P36 and P18 with WGA and AAL lectins, respectively, indicated the presence of lactosaminyl structures with sialic acid termini in P36 and of fucosylated residues in P18. Isoelectric focusing showed that the two proteins consist of several polypeptides. Some of these polypeptides were recognised by both human and rabbit antibodies: the pl of these epitopes was around 5.5 for P36 and 8.3-10.3 for P18. Rabbit antibodies detected the corresponding proteins on the sperm heads of methanol-fixed and of live acrosome-reacted spermatozoa. Anti-P36 antibodies bound mainly to the equatorial segment. They reduced the binding and, consequently, the penetration of zona-free hamster oocytes by human spermatozoa. Anti-P18 antibodies gave more diffuse staining of the acrosomal and post-acrosomal regions and reduced sperm-oocyte penetration without a significant effect on sperm binding. These results suggest that P36 and P18 antigens located in different compartments of the sperm head may participate in the sperm-oolemma interaction. We are currently investigating the physiological role of these antigens by sequencing the proteins isolated from the gels.
Asunto(s)
Antígenos de Superficie/inmunología , Infertilidad Masculina/inmunología , Proteínas de la Membrana/inmunología , Proteínas/inmunología , Espermatozoides/inmunología , Animales , Antígenos de Superficie/aislamiento & purificación , Western Blotting/métodos , Membrana Celular/inmunología , Cricetinae , Electroforesis en Gel de Poliacrilamida/métodos , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting/métodos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Proteínas de la Membrana/aislamiento & purificación , Oocitos/fisiología , Proteínas/aislamiento & purificación , ConejosRESUMEN
Electrotitration curves (ETC) of a marker protein mixture, pH 2.5-5.65, and human pepsinogens were performed in an agarose gel, containing 2% acid carrier ampholytes, forming a pH range of 2.5-5. Although the establishment of the pH gradient by isoelectric focusing was not quite complete and linear, both biochemically and immunochemically different types of pepsinogen C (PGC) and pepsinogen A (PGA) zymogens as well as the acid isoelectric points (pI) marker proteins were separated with good resolution. Three main fractions of PGA (Pg3, Pg4, and Pg5) were detected. To obtain an exact determination of the pepsinogen pIs, a simple and very fast 10 s pressure blot technique was applied. Human pepsinogens were separated alone or mixed with pI marker proteins in the pH range 2.4-5.65. No effect of the markers was observed on the pepsinogen migration. To visualize the different protein samples in the gel and on nitrocellulose membrane, we have used colloidal gold (AuroDye) staining, proteolytic activity, and immunostaining with monoclonal antibodies anti PGA and PGC. The described method shows an ability to separate proteins at acidic conditions with a resolution comparable to isoelectric focusing with immobilized pH gradients, but much faster, easier, and cheaper. In addition, the technique allows us to determine precise and exact pI values, and is suitable for studies of the pepsinogen polymorphism and its role in gastric diseases.
Asunto(s)
Electroforesis/métodos , Pepsinógenos/análisis , Humanos , Concentración de Iones de Hidrógeno , Estómago/químicaRESUMEN
BACKGROUND: Type 1 hypersensitivity to natural rubber latex proteins is a well-recognized health problem. Recent data have shown that allergens can be extracted from natural latex mattresses. As Hev b 1 (rubber elongation factor) and Hev b 6.02 (hevein) were described as major allergens, the present work was carried out to evaluate their presence in latex mattresses as well as in latex gloves. METHODS: Extracted proteins from latex mattresses and gloves were separated by SDS-PAGE or two-dimensional gel electrophoresis, transferred onto nitrocellulose and detected with monoclonal antibodies specific for Hev b 1 and Hev b 6.02. RESULTS: The results showed that various forms of Hev b 1, as well as degradation products of Hev b 1 were detected in latex mattresses and gloves, whereas Hev b 6.02 was not detected either in mattresses or in gloves. In a standardized latex extract, Hev b 1 and Hev b 6.01 (prohevein) were identified by the monoclonal antibodies. CONCLUSION: The fact that only Hev b 1 was detected by immunoblot in latex articles indicates that Hev b 1 may be the last protein to be washed out of latex products and that the Hev b 1 content may be used as a criterion for the estimation of the allergenicity of the latex products.
Asunto(s)
Alérgenos/aislamiento & purificación , Lechos/efectos adversos , Látex/inmunología , Látex/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Antígenos de Plantas , Guantes Protectores/efectos adversos , HumanosRESUMEN
BACKGROUND: In some geographic areas birch pollen represents the most prominent cause for airborne allergic diseases. Up to 70% of patients allergic to birch pollen are hypersensitive to fruits, especially apples. Associations have been found, in some instances, with a sensitivity to aeroallergens and HLA class II genes. OBJECTIVES: We investigated whether susceptibility or resistance to birch pollen allergy with and without food allergy was associated with HLA class II genes. METHODS: Blood samples were obtained from 2 groups of unrelated European-born white adults: 42 atopic patients (31 of them with asthma) and 42 healthy control subjects with no personal or familial history of asthma or atopy. Their antibody responses to birch pollen, apples, grass, and weed pollens were evaluated by skin tests, RASTs, and immunoprints. Genomic DNA was extracted from PBLs. The exons of DQA1, DQB1, DRB1, and DPB1 genes were selectively amplified by using the PCR method. Genotyping was carried out by digestion of the amplified DNA products with allele-specific endonucleases (PCR-RFLP), which recognize allelic variations in the polymorphic exon. RESULTS: We found no significant differences in the frequency of DPB1 alleles between patients and control subjects. HLA class II DR4 and/or DR7 alleles were present in 42.6% of the patients and in only 2.4% of the healthy subjects. These results confirm a previous study of a group of polysensitized atopic patients, which showed that DR4 and DR7 alleles were rare in healthy control subjects and frequently observed in atopic subjects with or without concomitant asthma. CONCLUSION: We conclude that the allele HLA-DR7 is significantly involved in the presentation of apple and pollen allergens. However, we suggest that this susceptibility is more related to atopy than to specific responses to allergens.
Asunto(s)
Alérgenos , Hipersensibilidad a los Alimentos/genética , Hipersensibilidad a los Alimentos/inmunología , Antígeno HLA-DR7/genética , Proteínas de Plantas/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Adulto , Alelos , Formación de Anticuerpos , Antígenos de Plantas , Femenino , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad Clase II/genética , Prueba de Histocompatibilidad , Humanos , Hipersensibilidad Inmediata/genética , Masculino , Rinitis Alérgica Estacional/genéticaRESUMEN
BACKGROUND: Latex allergy is a well-recognized health problem. The wide use of latex in daily life raised the question whether latex articles could be a source of allergens. The present study was carried out to analyse various latex mattresses for protein and allergen characterization. METHODS: Five latex foam mattresses were reduced to powder and proteins were extracted using either water or urea. The protein content of the extracts was analyzed by SDS-PAGE, IEF, 2-D gel electrophoresis and amino acid sequence analysis. The presence of specific IgE, directed toward mattress proteins found in human sera of latex-allergic patients, was tested using ELISA and immunoprints. RESULTS: The results showed that no protein or allergen could be extracted from synthetic foam. However, depending on the type of mattress and extraction procedure, various quantities of proteins could be extracted from mattresses containing natural latex. The ELISA levels of various latex-allergic patients were always more significant against urea extracts than against water extracts, except for one mattress. The protein and allergen patterns were qualitatively similar for all mattresses containing natural latex. Hev b 1 was identified by N-terminal amino acid sequence analysis. CONCLUSION: Because the natural rubber of the mattresses contains latex allergens, these allergens are a potential source of sensitization and could constitute a risk, at least to allergic individuals.
Asunto(s)
Inmunoglobulina E/inmunología , Hipersensibilidad al Látex , Alérgenos/inmunología , Lechos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Inmunoglobulina E/sangre , Hipersensibilidad al Látex/sangre , Hipersensibilidad al Látex/inmunología , Masculino , Proteínas/inmunologíaRESUMEN
BACKGROUND: Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized. OBJECTIVE: This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis. METHODS: D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced. RESULTS: 2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus. CONCLUSION: 2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.
Asunto(s)
Alérgenos/química , Ácaros/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Dermatofagoides , Quitinasas/química , Electroforesis en Gel Bidimensional , Glicoproteínas/química , Humanos , Técnicas de Inmunoadsorción , Focalización Isoeléctrica , Datos de Secuencia Molecular , Peso Molecular , Mapeo PeptídicoRESUMEN
This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Immunoblotting/métodos , Alérgenos/análisis , Alérgenos/inmunología , Colodión , Membranas Artificiales , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunología , Polen/inmunologíaRESUMEN
The genetic basis of allergic response to grass pollen allergens of 23 portuguese families have been studied. The two or three generations families formed a total of 128 individuals including at least one parent and one child sensitive to Dactylis glomerata grass pollen. HLA class II genes were studied by PCR amplification and RFLP analysis. Our population study has revealed a higher frequency of allele 2 of DOB in the allergic population than in non allergic individuals. 60% of cocksfoot sensitive patients are DR4 as compared to 20% in healthy population. Using the immunoprinting technique to study the specificity of the different immunoglobulin isotypes, we were able to improve the phenotyping quality. This analysis showed that patient IgG4 and IgE recognize frequently the same allergens. Some pollen allergens phenotypes (IgE) are transmitted from parents to children. Pollen specific IgG1 or IgG3 phenotypes might be better markers than IgA or IgM phenotypes to follow the natural immune response transmission in family studies.
Asunto(s)
Alérgenos/inmunología , Antígenos HLA-DR/genética , Proteínas de Plantas/inmunología , Polen/inmunología , Rinitis Alérgica Estacional/genética , Alérgenos/aislamiento & purificación , Genotipo , Antígenos HLA-DR/inmunología , Humanos , Hipersensibilidad Inmediata/genética , Hipersensibilidad Inmediata/inmunología , Immunoblotting , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Focalización Isoeléctrica , Fenotipo , Proteínas de Plantas/aislamiento & purificación , Poaceae , Reacción en Cadena de la Polimerasa , Rinitis Alérgica Estacional/etiología , Rinitis Alérgica Estacional/inmunologíaRESUMEN
Multiple successive pressure blottings of a single agarose isoelectric focusing gel were performed on normal and CNBr-activated nitrocellulose (NC) filters. The results obtained by multiple successive 10s immunoprints were compared to those obtained by a single 10 min immunoprint. To quantify the transfer efficiency of these techniques, a defined amount of radioactive material was separated by isoelectric focusing on agarose gel. After separation and pressure blottings of the gel the NC filters were submitted to autoradiography. The amount of radioactive material bound to the NC filters was determined by scintillation technique. The single 10 min pressure blot was more efficient than each of the multiple successive 10s prints. However, the latter procedure allowed equal resolution and resulted in a higher recovery of total radioactivity than the single immunoprint technique. The aim of this paper is to show how to obtain highly reproducible prints of electrophoretic patterns in an agarose gel of heterogeneous samples when accurate multiple immunodetections are to be performed. This technique was tested to characterize the grass pollen specific immunoglobulin classes and subclasses from an allergic patient.
Asunto(s)
Hipersensibilidad/inmunología , Immunoblotting , Inmunoglobulinas/análisis , Focalización Isoeléctrica , Polen/inmunología , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina G/análisis , Radioisótopos de YodoRESUMEN
Water-soluble grass pollen extracts (Dactylis glomerata) were separated by isoelectric focusing in a wide pH range (2-11) in agarose gel. After focusing, two successive gel prints were taken. The first one, on an ordinary nitrocellulose filter during 10 s, enabled the visualization of separated components of the pollen, after india ink staining. The second one, on a cyanogen bromide-activated nitrocellulose filter obtained after a 10-min transfer and saturation step, was incubated overnight with patient sera, and the specific antibodies bound to antigens or allergens were detected. The screening of different patient sera showed great variability in the antigen spectra. There was no obvious relationships between IgM and IgA antigen spectra as compared to IgE. Some association between IgE and IgG4 subclass was observed.
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Alérgenos/inmunología , Hipersensibilidad/inmunología , Poaceae/inmunología , Polen/inmunología , Alérgenos/análisis , Especificidad de Anticuerpos , Western Blotting , Humanos , Inmunoglobulina A/clasificación , Inmunoglobulina A/inmunología , Inmunoglobulina E/clasificación , Inmunoglobulina E/inmunología , Inmunoglobulina G/clasificación , Inmunoglobulina G/inmunología , Isotipos de Inmunoglobulinas/clasificación , Isotipos de Inmunoglobulinas/inmunología , Inmunoglobulina M/clasificación , Inmunoglobulina M/inmunología , Punto Isoeléctrico , Polen/aislamiento & purificaciónRESUMEN
Conventionnal (C) trouts and trouts obtained by gynogenesis (G) or self-fertilization (SF) were immunized with DNP-KLH and anti-DNP antibodies were individually purified by affinity chromatography. The isolated IgM-like antibodies were separated by an iso-electrofocusing technique in reducing conditions and electroblotted onto nitrocellulose. The transfers were probed with a mouse monoclonal antibody specific for trout heavy (H) antibody chain and revealed with a rabbit anti-mouse IgG horse-radish peroxydase conjugate. When comparing the IEF H chain spectrotypes of C, G and SF trouts, it was observed that individual C spectrotypes are more different between themselves than G and SF spectrotypes, and that individual SF spectrotypes were less heterogeneous than C or G ones. These results suggest that in trout, inbreeding induces a reduction of antibody diversity and heterogeneity. The inheritance of antibody repertoire might be taken in account in the inbreeding selection schedules for fish of economical interest.
Asunto(s)
Diversidad de Anticuerpos , Cadenas Pesadas de Inmunoglobulina/análisis , Salmonidae/inmunología , Trucha/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Dinitrobencenos/inmunología , Fertilización , Cadenas Pesadas de Inmunoglobulina/genética , Focalización Isoeléctrica , Xenopus/inmunologíaRESUMEN
The anti-DNP antibody responses were compared between conventional trouts produced in fishfarm and trouts experimentally obtained by self-fertilization of hermaphrodites or by gynogenesis. The variability of anti-DNP antibody titres was equivalent between normal and gynogenetic fish, but significantly reduced for fish obtained by self-fertilization. Isoelectrofocusing analysis of individually-isolated anti-DNP antibodies indicate low molecular heterogeneity but large variability between conventional trouts. Almost complete sharing of individual spectrotypes was observed between self-fertilized fish and large similarities between gynogenetics animals. These results suggest that the intensity of antibody synthesis in trout is under a complex genetic control, even in inbred animals expressing large similarities in their antibody repertoires.