Rapidly released allergens from short ragweed pollen: II. Identification and partial purification

We have identified several basic allergens in 16-min extracts of short ragweed (Ambrosia elatior) pollen and obtained a partially purified fraction (G50 II) using a combination of ion-exchange (CM-Sephadex C25) and gel-filtration (Biogel P30 and Sephadex G50) procedures. G50 II was allergenically and antigenically distinct from known allergens antigen E, Ra3, and Ra5. It gave a highly symmetrical peak on Sephadex G50 (fine), corresponding to a molecular weight of 11,500 daltons, and a single band on SDS polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. However, two cathodic antigens were detectable by both agarose electrophoresis and crossed immunoelectrophoresis. Allergenic activity of G50 II, assessed by puncture testing in ragweed-sensitive subjects, showed a characteristic pattern of response independent of patient response to AgE, Ra3, and Ra5. The major antigenic and allergenic component of G50 II is designated Ra6.     

Isolation and identification of pollen allergens of Artemisia scoparia

The allergenic proteins of Artemisia scoparia pollen were separated and identified with ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and RAST-inhibition techniques. The important allergenic component Artemisia VI b that constitutes 29% of total protein in the extract was purified to homogeneity. It was found to be an acidic protein with isoelectric point 3.8 and molecular weight of 14,300. It was rich in carbohydrate, but the carbohydrate portion did not appear to be important for allergenicity. In the crossed immunoelectrophoresis reference pattern of the whole pollen extract, 37 precipitin lines could be identified on the anodic side, whereas Artemisia VI b could be observed as a single precipitin line. Immunologically, the whole pollen extract of A. scoparia demonstrated shared antigenic and allergenic determinants with Ageratum conyzoides-pollen extract. The use of fast protein liquid chromatography in partial purification of allergenic components is also discussed.     

艾蒿花粉主要变应原的分离、纯化与鉴定

目的　对我国蒿属花粉中常见、重要的变应原艾蒿花粉进行分离、鉴定与纯化。方法采用不同的提取液得到艾蒿花粉粗浸液 ,经饱和 (NH4 ) 2 SO4 分级沉淀后用聚丙烯酰胺凝胶电泳 (SDS PAGE)分离蛋白质组分 ,并用凝胶成像系统测定各组分的相对分子质量 (Mr) ;采用Westernblot鉴定其主要及次要变应原 ;通过DEAE CelluloseDE 32离子交换层析 (ionexchangechromatography ,IEC)和SephadexG 75凝胶层析 (gelchromatography)对艾蒿花粉变应原进行纯化。结果　分离后得到 2 0多种蛋白质组分 ,其中Mr 为 5 8× 1 0 3、38× 1 0 3、2 5× 1 0 3、2 0× 1 0 3、1 6× 1 0 3等 5个条带蛋白含量最丰富 ;分离到的蛋白质组分中有 9种蛋白能与确诊的蒿属花粉过敏患者血清中蒿属花粉特异性IgE结合 ,其中Mr 为 6 2× 1 0 3、4 3× 1 0 3、38× 1 0 3的蛋白条带的结合率最高 ;经纯化后仅得到Mr 为 6 2× 1 0 3的主要变应原。结论　艾蒿花粉的主要变应原Mr 分别为 6 2× 1 0 3、4 3× 1 0 3和 38× 1 0 3,层析技术可以对Mr 为6 2× 1 0 3的主要变应原成分进行纯化。     

Identification of Parietaria judaica pollen allergens

Parietaria judaica pollen allergens, fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, were identified using 52 sera collected in Australia and Sicily from P. judaica pollen-allergic patients. IgE-binding pollen components transferred to nitrocellulose were detected by reaction with 125I-anti-human IgE and autoradiography. Nine pollen components, ranging in molecular weight (MW) from approximately 10,000 to 80,000 daltons, bound IgE antibodies but the two fastest migrating components sometimes each separated into two very closely migrating bands. The faster of the two components exhibiting doublet formation (MW approximately 10,000 daltons) showed by far the highest frequency of IgE binding, being recognised by 50 of the 52 sera examined. Although patients' IgE reaction patterns to P. judaica allergens were heterogeneous, the degree of heterogeneity was much less than that observed with house dust mite and other pollen extracts studied by electrophoretic transfer analysis. Results with gradient gel-nitrocellulose transfer experiments which showed no IgE-binding components with MWs less than 70,000 daltons, and comparisons of our electroblotting results with crossed radioimmunoelectrophoresis results of others, suggested that the doublet proteins with MWs of approximately 10,000 probably bind to higher MW proteins in P. judaica pollen extracts.     

Isolation and partial characterization of allergens from Helianthus annuus (sunflower) pollen

We have purified four allergens from Helianthus annuus (sunflower) pollen, hereafter named as allergens a, b, c , and d. Under native conditions, allergen a has a mol. mass of 32, allergen b has one of 24, and allergens c and d each have one of 55 kDa. At the least, allergens b , c, and d demonstrate charge heterogeneity, and the electrophoretic mobility of allergens c and d increases when these allergens are deglycosylated with trifluoromethanesulfonic acid. Cross-reactivity among the four allergens and with the whole extract is very high, and each allergen recognizes IgE in a high proportion of patients sensitized to sunflower pollen.     

The allergens of dog II. Identification and partial purification of a major dander allergen

A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, using sera from 32 dog-allergic subjects, a number of IgE radio-staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21 000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size-exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21 000 and an isoelectric point of approximately 5.2. An additional protein-staining band could still be seen of MW of 24 000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio-immunoelectrophoresis (CRIE) and that Blands et al. [2] and Løwenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.     

Identification of cross-reactive and genuine Parietaria judaica pollen allergens

BACKGROUND: The weed Parietaria judaica is one of the most important pollen allergen sources in the Mediterranean area. OBJECTIVE: We sought to identify P judaica pollen allergen, which might be used to serologically distinguish genuine Parietaria sensitization and cross-reactivity to allergens from other weed species (eg, mugwort and ragweed). METHODS: The allergen profile of P judaica IgE-reactive sera from weed pollen-sensitized allergic individuals from the Mediterranean region (n = 36) with high Parietaria pollen exposure and from weed pollen-allergic patients with little or no Parietaria exposure (Austria, n = 42; Scandinavia, n = 8; United States, n = 19) was established by CAP FEIA measurements and by IgE immunoblot inhibition experiments with recombinant allergens. RESULTS: The majority (83%) of the Mediterranean weed pollen-allergic patients mounted high IgE antibody levels (mean specific IgE, 20.89 kUA/L) against recombinant (r) Par j 2, whereas only 7% of the non-Mediterranean weed-allergic patients showed low IgE reactivity to rPar j 2 (mean specific IgE, 1.03 kUA/L). The cytoskeletal protein profilin and a 2-EF-hand calcium-binding allergen were identified as cross-reactive Parietaria allergens, which were recognized preferentially by Parietaria -positive, non-Mediterranean weed pollen-allergic patients. CONCLUSION: rPar j 2 might be used as a diagnostic marker allergen to identify weed pollen-allergic patients who are genuinely sensitized against Parietaria pollen and thus would be particularly suited for specific immunotherapy with Parietaria pollen extract.     

Identification and characterization of important allergens from mugwort pollen by IEF, SDS-PAGE and immunoblotting

Mugwort (Artemisia vulgaris L.) pollen allergens, separated by SDS-PAGE or IEF, were identified after transfer to NCM by incubation with a panel of sera from 16 patients with clinical mugwort pollen allergy, followed by [125I]anti-IgE and autoradiography. Of the at least 23 components separated by SDS-PAGE in a 15% polyacrylamide gel, at least 15 components with mol. wts 12,000-100,000 bound IgE from the panel of patient sera. A component of mol. wt 22,000 bound IgE from at least 94% of the patient sera tested and for all but three sera this component also bound the greatest quantity of IgE. Five other components with mol. wts 12,000, 17,000, 29,000, 39,000 and 42,000 bound IgE from 75-94% of the patient sera. After separation by IEF, at least 28 protein bands were detected in the pI region 3.5-7.2 and at least seven bands were found in the region 8.6-9.3. At least 11 bands in the pI range 4.2-7.3 and at least five bands in the pI region 8.5-9.2 bound IgE from the panel of patient sera. The most intense radiostaining was observed with a component having a pI of 4.35, which bound IgE from 31% of the patient sera. Immunoblotting of the SDS-PAGE and IEF gels using specific rabbit antisera and human sera against three important mugwort pollen allergens, denoted Ag 9, Ag 12 and Ag 13, was performed to determine the mol. wt and pI of these allergens which had earlier only been identified in CIE/CRIE. The results revealed that Ag 13 had a mol. wt of 61,000 and a pI of 4.35, Ag 12 had a mol. wt of 22,000 and AG 9 had pIs in the region 4.55-5.55 (six isoforms). Ag 9 did not bind IgE after SDS-PAGE and was thus not identified in the SDS-PAGE pattern, and Ag 12 failed to be detected in the NCM after transfer from IEF gels. By crossed immunoelectrofocusing, Ag 12 was found to consist of several isoforms predominantly located in the pI region 3.5-5.1. The immunoblotting analysis also revealed that the glycoprotein allergen Art v II was not detected after transfer from either SDS-PAGE or IEF gels. In conclusion, immunoblotting analysis of SDS-PAGE and IEF gels are useful methods for characterization of mugwort pollen extract, but it should be noted that some important allergens which are easily identified in CIE/CRIE may fail to be detected by these methods.     

Nondiffusional release of allergens from pollen grains of Artemisia vulgaris and Lilium longiflorum depends mainly on the type of the allergen

BACKGROUND: Upon contact with a wet surface, mature pollen grains hydrate and release proteins including allergens. Knowledge of the release mechanism of allergens that are mainly localized intracellularly may allow the design of strategies for inhibition of allergen release and the consequent sensitization process. METHODS: An improved pollen chromatography was performed with Artemisia vulgaris and Lilium longiflorum pollen. Using three elution media of different pH, osmolality and salt concentration mimicking various types of wet surfaces, the time-dependent elution profiles of total protein, a cell wall-bound acid phosphatase activity (acPase), allergenic (profilin, Art v 1) and nonallergenic molecules (14-3-3 protein, actin) were monitored. RESULTS: The release kinetics of total protein and cell wall-bound acPase followed an exponential decrease in both pollen species indicating a diffusion-based protein release, whereas the elution profiles of profilin, Art v 1 and 14-3-3 protein showed nondiffusion characteristics. No general dependence on pH, osmolality or salt concentration of the elution media was observable in the elution profiles. Under the applied conditions, actin was not released indicating that the pollen grains remained intact during the elution. CONCLUSION: The elution profiles of pollen allergens indicated that substantial amounts of these proteins do not diffuse from the cell wall or are released from intracellular compartments during imbibitional leakage. Instead, a mechanism seems to operate that involves translocation from the pollen cytoplasm to the extracellular environment by crossing an intact plasma membrane. Such a mechanism would probably allow the use of pharmaceuticals for inhibition of allergen release.     

Cypress pollen allergy. Identification of allergens and crossreactivity between divergent species

Abstract. Studies employing sera from 34 subjects allergic to while cypress pine ( Callitris glaucophvlla ) pollen identified 18 IgE antibody-binding components in the pollen of this species, five of which (MWs∼94, 68, 64, 43 and 34 kDa) were recognized by all of the sera. Protein blotting and quantitative inhibition studies revealed clear cross-reactivity between C. glaucophylla and Cupressus sempervirens pollen proteins and striking similarities in the IgE recognition band patterns of the two pollens. Inhibition experiments with other pollen extracts revealed that sera from C. glaucophylla pollen-allergic subjects can be divided into two groups – those inhibited only by extracts from the two Cupressaceae pollens and those inhibited both by these pollen proteins and by pollen extracts from other species. Most of the crossreactions in the latter group cannot be explained on the basis of taxonomic relationships or separate sensitizations. As with previous studies on birch and olive pollens, we conclude that pollen allergenic crossreactivity is much more wide-ranging than generally believed.     

Biological standardization of pollen allergens from India.

Standardization of allergens are achieved by in vitro and in vivo methods. Some of the allergens from Western countries are standardized using biological potency of the extracts but no attempt has been made till now to standardize any of the pollen extracts from India based on biological units. Therefore, we have attempted to standardize two important pollen allergens Ricinus communis and Holoptelea integrifolia by biological methods. Broadly the methods adopted by Dreborg and Grimmer (1983) was followed. Skin prick tests were carried out with the extracts of R. communis and H. integrifolia on 15 allergic patients in five three fold log dilutions starting with 1:10, in 50% glycerinated buffer. Glycerinated buffer (50%) and histamine dihydrochloride (1 mg/ml) were used as negative and positive controls, respectively. The mean wheal diameter obtained with different concentrations showed a gradual systematic fall with increase in dilution. The mean relative diameter (% of histamine reaction) varied from 124.1 +/- 8.9 to 33.7 +/- 6.1 and 78.9 +/- 5.5 to 21.4 +/- 3.8 with the highest and lowest concentrations of R. communis and H. integrifolia pollen antigens, respectively. The histamine equivalent concentration of antigen 1,000 Biological Units (BU) obtained for crude pollen extracts of R. communis and H. integrifolia was 1:17 and 1:22 respectively.     

河虾主要过敏组分的分离、纯化、鉴定及其致敏性分析

目的:鉴定河虾中的过敏原组分,对主要过敏原组分进行纯化并分析其致敏性强弱。方法:用磷酸盐缓冲液(PBS)制备河虾蛋白粗提液,将其与11例虾过敏症患者血清IgE进行Westernblot,鉴定各种分子量的河虾过敏原组分;河虾蛋白粗提液用硫酸铵沉淀、G-50凝胶层析和阴离子交换层析等方法纯化其主要过敏原组分,再用虾过敏患者血清IgE进行Westernblot鉴定;并将纯化的相对分子质量(Mr)为21000、36000、800003种主要过敏原组分与虾过敏症患者血清IgE做间接ELISA,以分析其致敏性强弱。结果:West-ernblot结果显示,河虾蛋白粗提液出现9个阳性条带;其中3种主要过敏原组分21000、36000、80000与患者血清的反应率为36.4%,63.6%,45.5%;将这3种Mr的蛋白组分分别做间接ELISA,结果显示21000、36000、80000过敏原组分与11例虾过敏症患者的混合血清IgE结合的吸光值都显著高于河虾蛋白粗提液。结论:河虾中至少存在9个过敏原组分;21000、36000、8000等3种蛋白组分为河虾的主要过敏原组分,其中以36000过敏原组分致敏率和致敏性最强。进一步研究将探明21000、36000、80000等3种河虾过敏原组分的共同抗原表位,以期为明确食物过敏原检测、临床诊断和虾过敏原疫苗设计提供基础。     

Structural characterziation of pollen allergens

The molecular characterization of allergens has accelerated significantly since the wide-spread implementation of modern analytical methods. The combination of gene cloning and heterologous protein expression has generated an extensive array of allergens that is available for comparative analysis, as well as clinical applications. Several internet-accessible allergen databases integrate the accumulated information from biomedical research and clinical practice. Innovations in classical biophysical methods, such as mass spectrometry, X-ray crystallography, and nuclear magnetic resonance spectroscopy, have rendered complex biological macromolecules amenable to detailed structured analysis. The modern scientific era has realized the synthesis of bioinformatics, molecular biology, biochemistry, biophysics, and immunology, and given us the means needed to decipher the remaining mysteries of allergies. This article addresses how the synergism of modern scientific techniques has hastened our understanding of allergies, how these techniques are applied in the identification and characterization of allergens, and how these methods assist the radional development of clinical tools for allergy diagnosis and treatment.     

Identification, quantitation, and purification of cockroach allergens using monoclonal antibodies

A panel of murine IgG monoclonal antibodies (MAbs) was raised against German cockroach (CR) (Blattella germanica) extract and selectively screened to identify MAb directed against allergen(s) recognized by IgE antibodies. Sera from 28 CR-allergic patients were used as sources of IgE antibodies to detect allergens "presented" by the MAb. Four clones (10A6, 3G12, 8F4, and 1D4) produced MAb to allergen(s) that bound IgE antibodies. Quantitative radioimmunoassays were used to compare levels of the MAb-defined allergens in CR extracts. MAb 10A6 reacted with a cross-reacting allergen that was detected in 9/14 CR species, including Blattella, Periplaneta, Blatta, Leucophea, and Supella spp, at concentrations of 100 to 10,000 U/ml. In contrast, MAb 3G12, 8F4, and 1D4 were Blattella specific. The allergen defined by MAb 8F4 was purified by MAb affinity chromatography and size-exclusion by high-performance liquid chromatography. It is a 36 kd heat-sensitive protein, isoelectric point, 5.2 to 5.4. Allergen 10A6 was partially purified by isoelectric focusing and high-performance liquid chromatography. It is a heat-stable, acidic protein (isoelectric point 3.15). Based on comparison of their properties with properties of previously described CR allergens, the allergens defined by MAb 10A6 and 8F4 have been provisionally designated Blattella germanica allergen I (Bla g I) and Blattella germanica allergen II (Bla g II), respectively. Assays of six commercial CR skin test extracts demonstrated a 200-fold difference in Bla g I levels (4.7 to 1085 U/ml) and only two extracts that contained detectable Bla g II (248 and 324 U/ml). The results demonstrate that MAb can be used to identify and define CR allergens and that the strategy of the use of MAb as a first step in allergen analysis and purification can be very effective, especially for poorly characterized allergen extracts.     

Cross-reactivity between pollen allergens from common Pooideae grasses and rye

In Germany, specific pollen extracts are currently used for immunotherapy of rye ( Secale cereale ) allergy. Like most common grasses, S. cereale belongs to the Pooideae subfamily. Using sera from grass pollen-allergic patients, immunoblotting and ELISA inhibition studies demonstrate that a five-grass allergenic extract from Anthoxanthum odoratum , Dactylis glomerata, Lolium perenne, Poa pratensis and Phleum pratense inhibits over 90% of IgE binding to rye allergens. This result confirms the high degree of homology between allergens from S. cereale and common grass species. We conclude that a five-grass pollen mixture is appropriate for desensitization of patients who are allergic to rye pollen without the need for additional rye pollen extracts.