RESUMO
Modern approaches to the standardization of allergens require the use of not only biological methods, but also a wide range of physicochemical and immunological ones. To obtain Soviet standard samples, the optimum spectrum of methods for their characterization, evaluation and comparison with international standard samples should be definitely selected. After the preliminary study of a number of allergens by different immunochemical methods a set of such methods, including different kinds of electrophoresis, isoelectrofocusing, immunoblotting, etc., are proposed, these methods being considered obligatory for obtaining Soviet standard samples of allergens.
Assuntos
Alérgenos/química , Alérgenos/normas , Animais , Fenômenos Químicos , Físico-Química , Contraimunoeletroforese , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoquímica , Focalização Isoelétrica , Pólen/imunologia , Coelhos , Padrões de Referência , U.R.S.S.RESUMO
We compared our in-house reference extract (RE) and a production extract (PE) with the international reference preparation (IRP) of International Union of Immunological Societies of timothy-grass pollen, using various biochemical and immunochemical methods. Furthermore, we compared the IgE composition of our in-house grass-pollen serum pool (West Germany) with the serum pool recommended by the World Health Organization, using the RE for crossed radioimmunoelectrophoresis (CRIE). Our extracts (RE and PE) were nearly comparable with the IRP. Only by CRIE one more allergen could be detected in RE and PE than in IRP and in another extract purchased from an American company. This finding may depend on the region where the source materials were harvested and which source materials were used for the preparation of the extracts. Furthermore, by high-performance liquid chromatography in the low-molecular-weight range, two distinct peaks could be detected in the nondiafiltered IRP, which were detected in the diafiltered RE and PE as traces only. The IgE composition of our in-house grass-pollen serum pool was comparable with the serum pool recommended by the World Health Organization, as detected by CRIE using RE.
Assuntos
Alérgenos/normas , Imunoglobulina E/análise , Poaceae/imunologia , Pólen/imunologia , Especificidade de Anticorpos , Humanos , Extratos Vegetais/normas , Padrões de Referência , Organização Mundial da SaúdeRESUMO
Two different lots of Russian-thistle extracts from five commercial sources were evaluated by isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblots of Immobilon transfers from these separations. The immunoblots were developed by an enzyme-linked method and scanned with a densitometer. The two lots of a standardized extracts produced scans that suggested uniformity in allergenic components. Several of the nonstandarized extract lots also appeared quite comparable. With further refinement, immunoblotting with densitometric analysis appears to offer an attractive way to standardize allergen extracts.
Assuntos
Alérgenos/normas , Extratos Vegetais/normas , Densitometria , Eletroforese em Gel de Poliacrilamida , Humanos , Immunoblotting , Focalização IsoelétricaRESUMO
The reliability of prick testing with allergen preparations produced in Yugoslavia was assessed by comparison to the internationally accepted ones (Pharmacia Diagnostica) of 100,000 BU activity. The following allergens were used: the mite Dermatophagoides pteronyssinus in 107 subjects, and grass pollens: Dactylis glomerata, Poa pratensis, Phleum pratense and Secale cereale in 39 subjects. The clinical significance of the response was assessed in relation to the skin reaction to the negative control solution and positive histamine control (in concentration of 1 mg/ml). The results indicate that the allergen extract of Dermatophagoides pteronyssinus (3000 PNU/ml) produced in Yugoslavia does not provoke skin reactions comparable to those provoked by the standardized extract of 100,000 BU in contrast to the grass pollen allergens, with the exception of Poa pratensis, which evokes equivalent skin reactions. Thus grass pollen allergens are reliable extracts and can be applied with good confidence in routine work. The need to set more rigorous criteria for assessing skin prick reactions, when domestic, unstandardized products are used, emerged as a result of this study.
Assuntos
Alérgenos/normas , Ácaros/imunologia , Pólen/imunologia , Testes Cutâneos/normas , Alérgenos/imunologia , Animais , Humanos , Poaceae/imunologia , Testes Cutâneos/métodosRESUMO
Skin prick tests (SPTs) represent one of the most common techniques of skin testing and are of great value, especially when standardized extracts are used. Phazet is a puncture test involving needles coated with standardized allergens. Phazet and SPTs have been compared in 130 subjects tested with six standardized allergen extracts: orchard-grass, olive, and mugwort pollens, cat dander, Dermatophagoides pteronyssinus, and Alternaria. Patients have been carefully selected on the basis of a suggestive clinical history, SPTs, and RAST. For all allergenic species, at least 20 nonsensitive individuals have been tested. The sensitivity (true positive in percent of patients with disease) of Phazet was 99.4%, and its specificity (true negative in percent of patients without disease) was 99.2%. It is concluded that Phazet and SPTs with standardized extracts are equally effective in the diagnosis of immediate-type allergy and that standardized extracts have improved the diagnosis of allergy, especially for previously poorly characterized extracts, such as Alternaria.
Assuntos
Alérgenos , Testes Cutâneos/instrumentação , Adolescente , Adulto , Alérgenos/normas , Animais , Gatos , Criança , Humanos , Hipersensibilidade Imediata/diagnóstico , Pessoa de Meia-Idade , Extratos Vegetais/normas , Pólen , Teste de Radioalergoadsorção , Testes Cutâneos/métodosRESUMO
Six candidate extracts of Lolium perenne (rye-grass) pollen have been studied in 6 laboratories using a variety of immunochemical and physicochemical techniques. Radioallergosorbent test inhibition, crossed immunoelectrophoresis, crossed radio-immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with immunoblot, thin-layer isoelectric focusing and enzyme-linked immunosorbent assay inhibition were used to evaluate each of the coded extracts. The source materials were also studied for identity and possible contamination by light microscopy. On the basis of these data, the Rye-Grass Working Party recommended to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies that the extract coded C be chosen as the candidate international reference preparation.
Assuntos
Alérgenos/normas , Lolium/imunologia , Poaceae/imunologia , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Colódio , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Alemanha Ocidental , Imunoeletroforese Bidimensional , Cooperação Internacional , Focalização Isoelétrica , Itália , Países Baixos , Extratos Vegetais/normas , Teste de Radioalergoadsorção , Padrões de Referência , Suécia , Reino Unido , Estados UnidosRESUMO
The biological activity of a partly purified, biochemically/immunochemically characterized mugwort pollen allergen preparation and crude pollen extracts of mugwort, goosefoot and English plantain was determined by means of skin prick test (SPT). The patient inclusion criteria with mugwort were a well-defined positive clinical history and a positive SPT. Symptoms related to goosefoot/English plantain pollens are difficult to define, as these weeds flower during the grass pollen season. Thus patients tested with these allergens did not fulfill the most important inclusion criterion for so-called biological standardization. To elicit a wheal of the same size as that produced by histamine 1 mg/ml required 100 to 10,000 times more material from these weeds, than from mugwort and other pollen allergen extracts investigated earlier. One thousand Biological Units/ml (BU/ml) corresponded to 8.3 micrograms dry weight (dw/ml) of the crude and 1.8 micrograms dw/ml of the purified mugwort pollen allergen preparation. Only 7/22 goosefoot-and English plantain-tested patients were positive at conjunctival or nasal challenge. All three weeds showed a similar composition with 5-10 allergens by CIE/CRIE analysis and 10-13 by immunoblotting analysis. One dominating allergen (approx. 15,000 d), could be identified for each weed species by protein gel blot after separation by SDS g-PAGE. There was no other explanation for the difference in biological activity than the criteria of selection. If there is no obvious clinical history, which is the main patient inclusion criterion in biological standardization, then additional criteria should be used.
Assuntos
Alérgenos/normas , Pólen/normas , Testes Cutâneos , Adolescente , Adulto , Idoso , Alérgenos/classificação , Alérgenos/imunologia , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoeletroforese Bidimensional , Imunoglobulina E/análise , Masculino , Pessoa de Meia-Idade , Teste de Radioalergoadsorção , Padrões de ReferênciaRESUMO
House dust represents a complex mixture of individual, clinically important allergens that includes insects, danders, and pollens. Characterization and standardization of each of these allergens will provide significant advancement in our understanding and treatment of allergic respiratory disease.
Assuntos
Alérgenos , Alérgenos/normas , Animais , Asma/etiologia , Gatos , Baratas/imunologia , Cães , Poeira , Hipersensibilidade Alimentar/etiologia , Fungos/imunologia , Humanos , Ácaros/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/etiologia , Esporos/imunologiaRESUMO
We have investigated dialyzed extracts (A-F) of short ragweed pollens (Ambrosia elatior) from six consecutive production years and four different companies by protein nitrogen units, high performance liquid chromatography, isoelectric focusing, SDS-PAGE, RAST inhibition, crossed radioimmunoelectrophoresis, and antigen E contents. Extract A showed lowest yields in all quantitative assays. Isoelectric focusing, SDS-PAGE, crossed immunoelectrophoresis, and crossed radioimmunoelectrophoresis showed comparable patterns for all extracts except for extract A. Apparently, the activity of short ragweed pollens depends on the year of production and/or the skill of the producer. These findings are in contrast to similar examinations of other pollen species.
Assuntos
Alérgenos , Pólen/imunologia , Alérgenos/análise , Alérgenos/normas , Carboidratos/análise , Nitrogênio/análise , Pólen/análise , Proteínas/análise , Padrões de Referência , Fatores de TempoRESUMO
In comparison with conventional allergen preparations immunologically characterized allergens were tested by skin-prick-tests for reactions. These results were compared with those of Rasttests.
Assuntos
Alérgenos/normas , Testes Intradérmicos , Testes Cutâneos , Fungos/imunologia , Humanos , Pólen/imunologiaAssuntos
Alérgenos/normas , Extratos Vegetais/normas , Alérgenos/imunologia , Alergoides , Ensaios Clínicos como Assunto , Relação Dose-Resposta Imunológica , Formaldeído/farmacologia , Humanos , Focalização Isoelétrica , Peso Molecular , Extratos Vegetais/administração & dosagem , Pólen/efeitos dos fármacos , Radioimunoensaio , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/terapia , Testes CutâneosRESUMO
Six different birch pollen extracts were analyzed by 20 laboratories for the standardization of birch (Betula verrucosa) pollen extracts used for diagnosis and specific therapy of patients with birch pollen allergy. The extracts were collected and delivered by the International Union of Immunological Societies, Allergen Standardization Subcommittee. One of the extracts, designated M, was proposed as an international standard (IS)-candidate of birch pollen extracts. The protein content of the IS candidate M was found to be 1.12 mg/mL, more than 2-fold higher than any of the other extracts analyzed. This preparation was among the extracts containing the highest number of protein components, as shown by isoelectric focusing, 28 lines, and by 11 precipitates in crossed immunoelectrophoresis. The allergenic reactivities were tested by crossed radioimmunoelectrophoresis (CRIE) and by radioallergosorbent test (RAST)-inhibition. In CRIE, the proposed IS (M) showed similar affinity for binding patients' IgE as the other extracts, as judged by the autoradiographic illustrations. Except for extract L, the values of RAST-inhibition for the rest were very similar. An IS extract should qualify for the criteria suggested for an optimal allergen preparation, containing minimal amounts of non-allergenic antigens and providing quantitatively and qualitatively all the allergenic proteins. The appropriateness of this selection seems unjustified in view of the above criteria.