Separation of Alternaria into protein and carbohydrate fractions with phenyl Sepharose

Portnoy, Jay M.; Pacheco, Freddy; Ballam, Yolanda; Barnes, Charles S.

doi:10.1016/0091-6749(91)90124-7

Cited by 5 publications

(7 citation statements)

References 12 publications

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“…Two bands at 25 kD and 36 kD cannot be explained by this reasoning, and might be due to impurities in anti-A It a I. Another possible explanation is non-specific binding of the rabbit IgG-antibodies to carbohydrates, which have been found to be an important fraction of Alternaria extracts [31,32], and which would not stain as protein. However, as demonstrated by different immunoelectrophoretic techniques, anti-A It a I appears to be highly specific for the 28 kD MW band, which has been shown to occur also as a band at 14 kD MW under reducing conditions [17].…”

Section: Sds-pagejwesternblotmentioning

confidence: 95%

Predominance of the major allergen (Alt a I) in Alternaria sensitized patients

Kleine‐Tebbe

Worm

Jeep

et al. 1993

Clin Experimental Allergy

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Thirty-nine patients sensitized to Alternaria were evaluated using titrated skin-prick test (SPT), histamine release studies (HR), inhibition of RAST and immunoblotting studies. To determine the relevance of the major allergen, Alt a I, specific rabbit antibodies against Alt a I and Alt a B were used. The antibodies were preincubated at different concentrations: (i) with the Alternaria allergen dose required for maximum response in the HR assay (10 BU/ml) and (ii) with the Alternaria antigen coupled to RAST paper discs (1000 BU/disc). Dose dependent inhibition of histamine release (n = 30, mean = 80% +/- 4%, IC30 = 0.69 micrograms/ml) and of RAST (n = 7, IC30 = 4.4 micrograms/ml) was found in all patients sensitized to Alternaria as indicated by allergen induced HR. The greater the response to Alternaria in HR, the higher the antibody concentrations necessary for inhibition (P < 0.05). Immunoblot experiments (n = 25) using SDS-PAGE showed in all cases IgE- and IgG binding at approximately 28 kD, which is the size reported for the major allergen, Alt a I. In two cases, slight IgE binding at 45 and 66 kD was also found, while in two other patients, only IgE binding at 66 kD was seen. Our findings emphasize the major importance of Alt a I in patients sensitized to Alternaria.

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Section: Sds-pagejwesternblotmentioning

confidence: 95%

Predominance of the major allergen (Alt a I) in Alternaria sensitized patients

Kleine‐Tebbe

Worm

Jeep

et al. 1993

Clin Experimental Allergy

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“…The characterized allergens of Alternaria are cytoplasmic proteins (Achatz et al 1995, Portnoy et al 1991, many of which have been characterised. These include molecules that are involved in amino acid translation (Alt a 6 and 12), folding (Alt a 3, Alt a 4) of protein configurations or metabolism (Alt a 10 and 11).…”

Section: Discussionmentioning

confidence: 99%

The effect of sunlight on allergen release from spores of the fungusAlternaria

Mitakakis

O'Meara²,

Tovey³

2003

Grana

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“…The extraction of EP antigens was carried out in ammonium bicarbonate buffer (125 m M , pH 8.0) containing 5 m M ethylenediaminetetra-acetic acid and phenylmethylsulfonyl fluoride (1 m M ) for 5 h at 4°C. The extract was dialyzed using the same buffer, lyophilized and stored at –70°C until analysis [14]. The protein content of EP extract was determined using a modified method of Lowry et al [16].…”

Section: Methodsmentioning

confidence: 99%

“…The clinico-pathological tests including blood analysis (total leukocyte count/differential leukocyte count), chest and paranasal sinus X-ray, pulmonary function test, urine and stool were conducted under the supervision of a specialist in allergy treatment (one of the authors). Intradermal tests were carried out with EP spore-mycelial extract (1:500 w/v) on the patients with nasobronchial allergy at the Clinical Research Center, V.P Chest Institute, Delhi, as described earlier [14, 15]. Phosphate-buffered saline (PBS, 0.1 M , pH 7.4) was used as negative control and histamine diphosphate (100 µg/ml) as positive control.…”

Section: Methodsmentioning

confidence: 99%

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Purification and Characterization of a Major Cross-Reactive Allergen from <i>Epicoccum purpurascens</i>

Bisht

Arora

Singh

et al. 2004

Int Arch Allergy Immunol

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Background:Epicoccum purpurascens (formerly nigrum) (EP), is a ubiquitous saprophytic mould found both indoors and outdoors. Several studies have reported sensitization to EP in 5–7 % of different populations worldwide. The diagnosis of mould allergy requires a standardized fungal extract that contains all its important allergenic proteins. The crude allergen extract from EP was standardized earlier, however none of its allergens have been purified. Methods: A major allergen from spore-mycelia extract of EP was purified using concanavalin A (Con A) Sepharose chromatography, gel filtration and electro-elution. The allergen isolated was characterized for its IgE-binding ability and cross-reactivity with five well-known allergenic fungi by ELISA and immunoblot. Results: A 33.5-kD glycoprotein allergen of EP, Epi p 1, was purified to homogeneity. All the EP allergic patients’ sera tested recognized this protein. Periodate modification of Epi p 1 showed partial loss in IgE binding while proteinase K treatment caused complete loss in binding to IgE. Dose-dependent inhibition in binding of rabbit anti Epi p 1 antibodies was obtained with Epi p 1, Aspergillus fumigatus, Alternaria alternata, Curvularia lunata, Cladosporium herbarum and Fusarium solani in ELISA. Rabbit antibodies to all the above five fungi recognized Epi p 1 in immunoblot, confirming that Epi p 1 shares common epitopes with the fungi tested. Conclusion: A major glycoprotein allergen of 33.5 kD was purified from EP which cross-reacts with other fungi. Hence this glycoprotein can be exploited to reduce the panel of allergen extracts used for therapy of mould allergy.

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Separation of Alternaria into protein and carbohydrate fractions with phenyl Sepharose

Cited by 5 publications

References 12 publications

Predominance of the major allergen (Alt a I) in Alternaria sensitized patients

Predominance of the major allergen (Alt a I) in Alternaria sensitized patients

The effect of sunlight on allergen release from spores of the fungusAlternaria

Purification and Characterization of a Major Cross-Reactive Allergen from <i>Epicoccum purpurascens</i>

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