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Background Prosopis juliflora (mesquite) is one of the major sources of pollinosis in tropical and semi-arid countries of the world. The present study was undertaken to purify and characterize a major cross-reactive allergen from this tree species. Materials and methods Mesquite pollen extract was purified using reverse-phase chromatography. Allergen characterization was done by electrophoresis, enzyme-linked immunosorbent assay (ELISA) and Western blotting. Clinical relevance of the purified protein was analyzed by in vivo (skin tests) and in vitro experiments such as ELISA, histamine release, peripheral blood mononuclear cells (PBMC) proliferation and cytokine assays. Cross-reactivity of purified protein with allergenic tree species and lima bean (food) was assessed by inhibition assays. Results A 66-kDa protein was purified from mesquite pollen extract using octadecyl silica resin. Purified protein recognized 90% of mesquite-sensitized patients in skin test and ELISA. It induced significant histamine release in allergic patients’ blood and interleukin-4 secretion in the PBMC culture supernatants. Inhibition assays suggested close allergenic relationship of this protein with Ailanthus excelsa, Cassia siamea, Salvadora persica pollen and Phaseolus lunatus (lima bean – an edible legume). Conclusions A 66-kDa major cross-reactive allergen was isolated from mesquite pollen using single-step purification procedure. The protein seems relevant for clinical applications in allergic disorders.

Commercial cockroach extracts for diagnosis and therapy show batch-to-batch variation. This study aimed to standardize Periplaneta americana extract based on major IgE binding components using hypersensitive patients' sera. Extracts were prepared in phosphate buffered saline (PBS) or NH 4 HCO 3 , from freeze-dried or 37 °C dried material and compared with commercial extracts by immunobiochemical methods. Cockroach positive patients' sera were collected after intradermal tests and specific IgE enzyme linked immunosorbent assay (ELISA). Allergenic proteins were identified by western-blotting and potency of extracts determined by ELISA-inhibition. Adult P. americana extract from freeze dried source material in PBS (PA extract) resolved into 45 protein bands and showed 22 IgE binding components with pooled patients' sera. It required 9–12 ng self-proteins for 50% ELISA-inhibition. Individual patients' sera identified 23, 28, 35, 38, 40, 49, 72, 78 and 97 kDa as major IgE binding components in PA extract. Nymph extract exhibited similar potency and protein profile to PA extract with 72 and 78 kDa proteins present in high intensities. Commercial extracts exhibited only 6–11 IgE reactive bands compared to PA extract and required 40 folds or more protein for 50% ELISA-inhibition. PA extract from freeze-dried source material seems a potent allergen preparation with 9-major IgE binding components. It can be referred to upgrade the quality of commercial extracts exhibiting low potencies due to poor quality source material, inadequate extraction procedures and improper storage.

Objective: Attachment to host matrix is an important provisory step for the institution of any fungal infection. The present study investigates the role of glycoproteins of Epicoccum purpurascens in host-fungal adherence. Methods:Epicoccum spore-mycelial extract was fractionated on a concanavalin A-Sepharose column. Three glycoproteins of 12, 17 and 33 kDa (Epi p 1) were electroeluted and checked for hemagglutination and hemagglutination inhibition. The monosaccharide content of the highly potent protein Epi p 1 was determined by high-performance anion exchange chromatography and pulsed amperometric detection. The interaction of Epi p 1 with mannose-binding lectin (MBL) leading to the activation of the complement system was studied by immunoblot, ELISA and ELISA inhibition techniques. Immunoblot and immunoblot inhibition were carried out with culture filtrate to determine the nature of Epi p 1. Results: 33 (Epi p 1)-, 17- and 12-kDa proteins were 58, 46 and 38 times more potent than crude extract in hemagglutination activity (HA). The HA of Epi p 1 was inhibited by N-acetyl glucosamine, glucose and laminin. Epi p 1 had a high mannose content, showed MBL binding in ion-dependent manner and caused complement activation. The protein was detected in culture filtrate and thus seems to play a significant role in fungal invasion. Conclusion: Epi p 1, an allergenic glycoprotein of E. purpurascens, is involved in host-fungal interactions through MBL.

Clinical observations suggest the presence of cross-reactive allergens. There is a need to identify these cross-reactive allergens to improve the treatment used for allergic disorders. The present study was aimed to identify and characterize a cross-reactive allergenic protein from fungi. Allergen extracts of various fungi viz. Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Curvularia lunata, and Epicoccum purpurascens showed GST enzymatic activity ranging from 0.765 to 1.004 Δ340 nm/min/µg where as activity of rGST was 1.123 Δ340 nm/min/µg. Immunoblot with GST antibodies showed a band of approximately 26 kDa in all these fungal extracts. Sera of fungal allergy patients showed the presence of IgE antibodies to GST. Rabbit antibodies raised against the fungal extracts reacted with rGST confirming the presence of GST-like protein in these extract. ELISA inhibition using GST antibodies revealed inhibition with C. herbarum, A. alternata, C. lunata, A. fumigatus, and E. purpurascens demons...

Epicoccum purpurascens (EP) is a ubiquitous saprophytic mould, the inhalant spores and mycelia of which are responsible for respiratory allergic disorders in 5-7% of population worldwide. The diagnosis/therapy of these disorders caused by fungi involves the use of standardized and purified fungal extracts. A 33.5 kDa glycoprotein, Epi p 1 released histamine from whole blood cells of EP allergic patients at a concentration of 50-ng protein. The high specific IgE values detected in EP hypersensitive sera indicated that Epi p 1 is capable of mediating type I hypersensitive reaction in predisposed individuals. It also showed protease activity by virtue of its dose dependent cleavage of serine protease specific synthetic substrate, N-benzoyl arginine ethyl ester hydrochloride (BAEE). The serine protease nature of Epi p 1 was confirmed by its N-terminal sequence (ADG/FIVAVELD/STY) homology to a subtilisin like serine protease. The protease activity of Epi p 1 may be responsible for making its way into the system of pre-disposed individuals through epithelial cell detachment and the histamine releasing ability by cross-linking of IgE antibodies on cell surface is the cause of its allergenic nature.

Background: Several proteins from Curvularia lunata have been identified as important fungal allergens. It will be worthwhile to study the functional aspects of these allergens. The present study aimed at purifying a major allergen and determining its biological function. Methods: Concanavalin A and Superdex 75 were used to purify Cur l 1 major allergen from C. lunata. Cur l 1 activity was determined qualitatively and quantitatively. Serine protease inhibitors and specific substrate was used to determine the biological function of the protein. Results: Concanavalin A-bound fraction showed five allergenic proteins, which on Superdex G-75 purification gave a homogenous Cur l 1 protein. Cur l 1 showed IgE reactivity with 80% of the C. lunata hypersensitive patient's sera indicating it to be a major allergen. It showed protease activity on different substrates. Cur l 1's amino terminal sequence, GLTQKSAPWGLGADTIVAVELDSY, showed homology with the alkaline serine protease precursor. Phenylmethylsulfonylfluoride, pefabloc, aprotinin and leupeptin inhibited 70–80% enzymatic activity of Cur l 1 and no inhibition was observed with ethylenediaminetetraacetic acid (EDTA). A dose-dependent hydrolysis of Nα-benzoyl-l-arginine ethyl ester-hydrochloride, a specific serine protease substrate was obtained with Cur l 1. Conclusion: A major glycoprotein allergen Cur l 1 was purified to homogeneity from C. lunata. Amino terminal sequence and biochemical assays identified it as a serine protease.

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.

Background:Fusarium solani (FS) is an important source of fungal allergen. A 45-kD major allergen of FS showed reactivity with patients’ sera sensitive to many fungi. Objectives: To purify and characterize a 45-kD common allergenic protein from FS, which may be useful for the diagnosis of and therapy for fungal allergy. Methods: FS culture filtrate extract was separated on SDS-PAGE; 45-kD protein was electroeluted and purified on C18 column using reverse-phase high-pressure liquid chromatography (rpHPLC). The purified protein was functionally and biochemically characterized by in vitro and in vivo methods. Results: The 45-kD protein showed a single peak on rpHPLC. The N-terminal amino acid sequence of this protein did not show homology to enolase or known fungal proteins. It showed cross-reactivity with Epicoccum nigrum, Curvularia lunata, Cladosporium herbarum and Alternaria alternata by ELISA and ELISA inhibition using rabbit antibodies raised against these fungi. IgE ELISA inhibition with patients’ sera positive to different fungi demonstrated allergenic cross-reactivity of the 45-kD protein with other fungal extracts. This 45-kD protein released a significant amount of histamine in FS-allergic patients. Conclusion: A cross-reactive 45-kD allergenic/antigenic protein was purified to homogeneity and characterized. It has prospects for use in allergen therapy.

Background Previous studies have identified Epicoccum nigrum (EN) as an important aeroallergen. Shared allergenicity among some fungi responsible for type I allergic disorders has been reported. Objective To study the cross-reactivity among different fungi and to identify immunoglobulin (Ig)G and IgE binding components shared between EN and 10 other fungi known to cause respiratory allergy. Methods Cross-reactivity studies were carried out by enzyme-linked immunoadsorbent assay (ELISA) and immunoblot inhibition using both rabbit antiserum raised against EN and pooled sera from patients' EN-positive skin test. Results A large number (82%) of EN-sensitive patients showed positive skin reactivity to other fungal extracts. ELISA inhibition revealed >50% inhibition in binding of EN-specific rabbit antibodies with Alternaria alternata , Curvularia lunata , Cladosporium herbarum , and Penicillium citrinum extract, whereas the other extracts showed only 20 to 40% inhibition. Rabbit antisera to A. alternata , C. herbarum , and C. lunata reacted with five to seven bands in EN, demonstrating the presence of shared antigens among these fungi. EN requires an amount of 100 ng for 50% IgE ELISA inhibition, whereas 175 ng of A. alternata , 160 ng of C. lunata , and 268 ng of C. herbarum and P. citrinum were required for the same. IgE immunoblot and immunoblot inhibition further revealed that 43-, 26-, and 17-kD allergenic bands were shared by EN and A. alternata , whereas the 80- and 37-kD bands were common to both EN and C. lunata . EN and C. herbarum shared 63- and 36-kD allergenic bands, whereas EN and P. citrinum shared the 34-kD band. Conclusion EN showed maximum cross-reactivity with A. alternata followed by C. lunata , C. herbarum , and P. citrinum. This information will be useful in treating EN-allergic patients.

Background:Curvularia lunata is an important fungus for respiratory allergic disorders. Previous studies indicated cross-reactivity of Curvularia with other fungi. However, the cross-reactive allergenic component (s) were not identified. The present work was carried out to study the shared allergenic components of C. lunata and others. Methods: Cross-reactivity studies were performed using pooled hypersensitive patient sera to C. lunata by ELISA, immunoblot, immunoblot inhibition and ELISA inhibition. Results: Many C. lunata sensitive patients showed positive skin test to five other fungi. Alternaria alternata exhibited maximum (68%) whereas Cladosporium herbarum showed the least (17%) skin reactivity. Immunoblots of fungal extracts with pooled sera showed common proteins. Fusarium solani and C. herbarum showed negligible IgE binding. IgE ELISA inhibition with C. lunata showed 92% inhibition whereas A. alternata and E. nigrum showed 84% and 63%, respectively. Immunoblot inhibition with self protein showed complete loss of IgE-binding activity. Proteins of 26, 31, 38, 45 and 50 kDa of C. lunata were inhibited by A. alternata and E. nigrum, whereas A. fumigatus inhibited 26, 45 and 50 kDa proteins. Conclusions: Significant allergenic cross-reactivity exists among proteins of C. lunata, A. alternata and E. nigrum. Proteins of 26, 31, 38, 45 and 50 kDa are shared allergens in these fungi.

Epicoccum nigrum (EN) is an important fungal allergen for nasobronchial allergy. Fungal extracts should contain all the relevant allergen components from spores, mycelium and culture medium for the purpose of allergy diagnosis and therapy. EN extract from spore-mycelial mass has been standardized, but the culture filtrate (CF) allergens of EN have not been studied as EN grows poorly in synthetic medium. The objective of the present study was to obtain a standard CF extract of EN by cultivating the source material in a modified semi-synthetic medium and to compare this with the EN cellular extract. Sabouraud's medium containing yeast extract (50 mg/l) was filtered using 10-kDa cut-off membrane and the lower molecular mass media components were used to cultivate EN. The CF obtained after removing the spore-mycelia was dialyzed to remove media components. The CF extract was characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. It was compared with EN spore-mycelial extract by enzyme-linked immunosorbent assay (ELISA), ELISA inhibition and by intradermal testing on allergy patients. The CF extract of EN resolved into 30 protein bands on SDS-PAGE. About 27 IgG bands were detected using anti-EN rabbit antibodies and 12 IgE bands by EN-sensitive pooled patients' sera. Periodate modification of CF proteins showed that the carbohydrate moieties are not important for IgE binding. Protein components of 26, 34 and 43 kDa were recognized as the major CF allergens. Three different batches of CF extract required 7.5-9 ng of self protein for 50% inhibition of binding to anti-EN rabbit antibodies in ELISA. Intradermal testing with CF extract showed comparable allergenic potency to standardized EN spore-mycelial extract, although it contained some allergenic proteins in higher amounts as compared to the spore-mycelial extract. In summary, the semi-synthetic medium has been suitably modified for obtaining EN CF antigens. This medium can be an important substitute for producing potent CF allergens of fungi that grow poorly in synthetic medium. The EN CF extract elicited good allergenic reactivity and may be used for allergy diagnosis along with spore-mycelial extract.

Background:Curvularia lunata is an important inhalant allergen. The present study was undertaken to investigate the shared IgG- and IgE-binding components among seven Curvularia species prevalent in the aerospora. Methods: Seven different Curvularia species were grown in a semisynthetic medium for 13 days. The extracts were analyzed by SDS-PAGE, immunoblot and ELISA/immunoblot inhibition using sera from C. lunata-positive patients and anti-C. lunata rabbit serum. Results: Different Curvularia species showed 11–19 protein bands on SDS-PAGE. Proteins of 12, 20, 31, 45, 53, 78 and 97 kD were present in all the species. Eight out of 98 nasobronchial patients exhibited positive skin tests to C. lunata and to at least five Curvularia species. ELISA using these sera showed IgE binding with Curvularia species. Immunoblot using pooled anti-C. lunata sera from patients showed 5–12 allergenic proteins. Proteins of 12, 31, 45, 53 and 78 kD showed IgE binding in Curvularia species. Antibodies against C. lunata detected 6–14 antigenic proteins on immunoblot. Proteins of 31, 45 and 53 kD showed IgG binding in all the species. Proteins of 31 and 53 kD showed complete IgE/IgG binding inhibition. IgE/IgG ELISA inhibition showed dose-dependent inhibition in Curvularia species. C. lunata extract required 0.17 and 0.11 µg of protein for 50% IgE and IgG inhibition, respectively. C. clavata and C. pallescens required 10 times more protein to exhibit the same inhibition and other species required similar protein levels as those required by C. lunata.Conclusions: A high degree of cross-reactivity was observed between C. lunata and the six other Curvularia species tested. C. lunata and C. senegalensis shared maximum allergenic and antigenic components.

Isolation of total RNA from plant materials has been difficult, due to the presence of complex organic substances and the associated pigmentation. In fact, there is a dearth of standardized protocols for isolating total RNA from pollens. To find a simple and reliable method for isolating total RNA from pollen, four methods, viz. phenol/SDS (PS), guanidine HCl (GH), tri-reagent (TR), and modified SDS-betaME (SB) were tested with fresh pollen of Ricinus communis (procured at -70 degrees C) and pollen dried at 30-37 degrees C. The quality and quantity of RNA was superior for the material processed at -70 degrees C. SB gave the highest RNA yield (2.35 mg/g, OD260/280 >2.0), compared to other methods. The results obtained by the SB method were found to be comparable with the widely used tri-reagent method. This was validated with other pollens of Imperata cylindrica and Xanthium strumarium. The yield obtained from graded amounts of pollen was consistent with SB, compared to the TR method. The RNA isolated by SB gave good quality mRNA for synthesizing cDNA. The SDS-betaME method is simple, efficient, and uses less expensive reagents. Hence, we recommend the modified SDS-betaME method for isolating total RNA from pollens.

Background: Grass pollens are known to induce type I allergic reactions in a large number of genetically predisposed individuals. Earlier studies have recognized Imperata cylindrica (Ic) pollen as an important source of aeroallergen which contained 7 IgE binding proteins in the MW range of 85–16 kD. Objectives: To isolate, purify and characterize a cross-reactive allergenic protein from Ic pollen extract for diagnosis and therapy of grass pollen allergy. Methodology: Ic pollen extract was fractionated using DEAE Sephadex A-50, Sephadex G-200 and Mono Q column. Allergenic activity of the fractions was checked by ELISA, skin tests, ELISA inhibition and immunoblot using sera of Ic-sensitive patients. A 67-kD protein was purified to homogeneity from Ic-VIII. The allergenic determinants of this protein were identified by SDS-PAGE and immunoblot after CNBr treatment. Results: Among Ic fractions, Ic-VIII was highly potent by ELISA, skin tests and showed cross-reactivity with 4 other tropical grasses by immunoblot and ELISA inhibition. The subfraction Ic-VIIIe1 of Ic-VIII showed a band at 67 kD on SDS-PAGE. On CNBr treatment, it gave 7 peptides, 3 of which were found to be allergenic. Conclusion: A 67-kD protein (Ic-VIIIe1) was isolated, purified to homogeneity and partially characterized. It showed cross-reactivity with tropical grasses tested and contained at least three allergenic determinants.

Putranjiva roxburghii (PR) pollen has been found to be an important aeroallergen for type I hypersensitivity. In the present study, the IgE binding proteins of PR pollen have been c

Brassica campestris (BC), Eng. Mustard, is an important source of pollen allergen, responsible for type I hypersensitivity disorders. In the present study, BC pollen extract was characterized by TLIEF, SDS-PAGE and immunoprinting. The extract separated into 50 silver stained bands of pI 3-9 on isoelectric focusing whereas it resolved into 14 Coomassie blue stained protein bands of 14-100 kD on SDS-PAGE. Immunoblot analysis with individual patient sera detected four allergenic proteins of 90, 67, 60 and 14 kD. BC separated into 8 peaks (Bras 1-8) on DEAE Sephadex A-50 column. Bras 2 was found to be most potent by IgE specific ELISA, hence further fractionated on Sephadex G-200. A protein of 90 kD (Bras 2a) isolated by gel filtration was found to be most allergenic protein by ELISA inhibition. The findings shall be applicable in standardization of future batches of BC pollen extract to be used for allergy diagnosis and immunotherapy.

Legumes are the major elicitors of IgE-mediated food allergy in many countries of the world. Purified major allergens are prerequisite for component resolved diagnosis of allergy. The present study was aimed to isolate and characterize a major allergenic protein from blackgram ( Phaseolus mungo ). Respiratory allergy patients with history of blackgram allergy were skin prick tested (SPT) and sera were collected from SPT positive patients. The blackgram extract was fractionated using a combination of anion exchange and hydrophobic interaction chromatography. The purified protein was characterized by indirect ELISA, immunoblot, ELISA inhibition, SPTs, stripped basophil histamine release, lymphoproliferation assay and digestibility assay. The purified protein separated at 28 kDa on 12% gel and showed IgE binding with 81% of blackgram hypersensitive patients’ sera on immunoblot indicating it to be a major allergen. Periodic Acid Schiff's and meta-periodate treatment staining detected it to be a glycoprotein. The 28 kDa protein recognized 7/9 (77.8%) of blackgram positive patients by SPT, where as all 9 patients showed significant histamine release on stimulation with protein as compared to controls. The 28 kDa protein remained stable up to 15 min on incubation with SGF. Bands of 14–16 kDa appeared after 15 min of pepsin digestion that remained stable up to 60 min of incubation. However, purified protein degraded within 5 min after incubation with SIF. The N-terminus-12 residues sequence of 28 kDa protein was GRREDDYDNLQL. A stretch of residues ‘DDYDNLQL’ showed homology with Rho-specific inhibitor of transcription termination ( E = 0.42, Identity = 87%) and NBS-LRR type disease resistant protein from peanut ( Arachis hypogaea ) ( E = 2, Identity = 77%). In conclusion, the purified 28 kDa protein is a potent major allergen that may have implication in diagnosis of blackgram allergy.

Exposure to Epicoccum purpurascens is implicated in respiratory allergies and asthma. Several allergens of clinical importance were identified in Epicoccum extract (EE), but only one allergen has been isolated and characterized. In the present study, a 12-kDa allergen was isolated from an Epicoccum spore-mycelial extract by concanavalin-A sepharose, reverse-phase hydrophobic and gel filtration chromatography. The purified protein was recognized as a single 12-kDa allergen on immunoblot with a serum pool of Epicoccum-sensitive patients. Of the 94 respiratory allergy patients tested intradermally, 17 showed marked positive skin reactions to EE and 12 of them reacted with the 12-kDa protein, indicating a diagnostic sensitivity of 70%. More than 80% patients' sera showed immunoglobulin E (IgE) reactivity to the purified protein in enzyme-linked immunosorbent assay and immunoblot, identifying it as a major allergen. Preincubation of pooled serum with the protein led to inhibition of IgE binding to solid-phase-bound EE (effective concentration 50%=180 ng). Twelve of the 17 serum samples showed significant basophil histamine release upon stimulation with purified protein. The protein induced significant proliferation of peripheral blood mononuclear cells of 13 patients. A high level of interleukin-4 in the culture supernatant of these cells indicated induction of a T-helper type 2 response. The purified 12-kDa protein is a clinically relevant allergen and has potential for the diagnosis and therapy of Epicoccum allergies.

Studies suggest the importance of serum total and specific IgE in clinical evaluation of allergic manifestations. Such studies are lacking in Indian subcontinent, though a large population suffer from bronchial asthma. Here relevance of serum total and specific IgE was investigated in asthmatics with food sensitization. A total of 216 consecutive patients (mean age 31.9 years, S.D. 11.8) were screened by various diagnostic testing. Out of 216 patients, 172 were with elevated serum total IgE (201 to800 IU/ml). Rice elicited marked positive skin prick test reactions (SPT) in 24 (11%) asthma patients followed by black gram 22 (10%), lentil 21 (9.7%) and citrus fruits 20 (9.2%). Serum total IgE and specific IgE showed significant correlation, p = 0.005 and p = 0.001, respectively, with positive skin tests. Blinded food challenges (DBPCFC) with rice and or black gram confirmed food sensitization in 28-37% of cases. In summary, serum total IgE of 265 IU/ml or more with marked positive SPT (4 mm or more) can serve as marker for atopy and food sensitization. Specific IgE, three times of normal controls correlates well with positive DBPCFC and offers evidence for the cases of food allergy.

Attachment to host matrix is an important provisory step for the institution of any fungal infection. The present study investigates the role of glycoproteins of Epicoccum purpurascens in host-fungal adherence.Epicoccum spore-mycelial extract was fractionated on a concanavalin A-Sepharose column. Three glycoproteins of 12, 17 and 33 kDa (Epi p 1) were electroeluted and checked for hemagglutination and hemagglutination inhibition. The monosaccharide content of the highly potent protein Epi p 1 was determined by high-performance anion exchange chromatography and pulsed amperometric detection. The interaction of Epi p 1 with mannose-binding lectin (MBL) leading to the activation of the complement system was studied by immunoblot, ELISA and ELISA inhibition techniques. Immunoblot and immunoblot inhibition were carried out with culture filtrate to determine the nature of Epi p 1.33 (Epi p 1)-, 17- and 12-kDa proteins were 58, 46 and 38 times more potent than crude extract in hemagglutination activity (HA). The HA of Epi p 1 was inhibited by N-acetyl glucosamine, glucose and laminin. Epi p 1 had a high mannose content, showed MBL binding in ion-dependent manner and caused complement activation. The protein was detected in culture filtrate and thus seems to play a significant role in fungal invasion.Epi p 1, an allergenic glycoprotein of E. purpurascens, is involved in host-fungal interactions through MBL.

Recombinant allergens are required to study allergy at the molecular level and are helpful tools for the improvement of diagnosis and therapy. In the present study, enolase was expressed from Curvularia lunata and analyzed for its immunological reactivity as an allergen. cDNA library was synthesized in lambda zap vector and screened with sera obtained from C. lunata allergic patients. A cDNA clone with an ORF of 1.3 kb showed homology to enolases from different fungal sources. It was expressed in E. coli, purified from inclusion bodies yielding 0.5 mg/L and showed enzyme activity of 48 units/mg. It resolved as 48-kDa band on SDS-PAGE and was recognized by all the individual Curvularia positive patient sera in immunoblot and ELISA. r Cur l 2 stimulated patients' PBMCs and supernatant of these cells showed elevated levels of Th 2 cytokines. Ten B cell epitopes were predicted using computational software and one showed 90% homology to an important IgE epitope of Cla h 6. The various parameters predicted by computational approach can be validated later as a future study to draw conclusive evidence about putative antigenic epitopes. This can further help in generating knowledge about residues important for IgE binding and developing therapeutic modalities.

Background: Legumes are important causative agents of type I hypersensitivity in south Asia and Europe but such studies are lacking in Indian population. The present study investigates blackgram sensitization in asthma and rhinitis patients and identifies immunoglobulin E (IgE)-binding proteins. Methods: Respiratory allergy patients were evaluated using standard questionnaire and skin prick tests (SPT) with common foods and aeroallergens. Blackgram-specific IgE level was estimated by enzyme-linked immunosorbent assay (ELISA) and sensitization was established by a double-blind, placebo-controlled food challenge (DBPCFC). The cross-reactivity of blackgram with other legumes was studied by immunobiochemical methods. Result: Of 816 patients, 35 gave history of blackgram hypersensitivity. From these, 16 patients were SPT positive and 14 showed elevated specific IgE (three times of negative control) to blackgram. DBPCFC established blackgram sensitivity in four of 14 patients. Immunoblotting with individual patient's sera recognized eight most prevalent allergens of 78, 56, 47, 43, 40, 30, 28 and 16 kDa. Roasted blackgram showed six major allergens whereas 47, 43 and 28 kDa proteins retained IgE reactivity upon boiling. Blackgram extract required 14 ng of self protein for 50% ELISA inhibition whereas roasted and boiled blackgram required 16 and 120 ng protein. ELISA and immunoblot inhibition show partial inhibition to blackgram proteins by lentil, limabean and pea. Conclusion: Blackgram induces IgE-mediated reactions in 1.7% of asthma and rhinitis patients and contains eight major IgE-binding components, of which six retained IgE reactivity after roasting. Blackgram shares allergenicity with lentil and limabean.

Clinical observations suggest the presence of cross-reactive allergens. There is a need to identify these cross-reactive allergens to improve the treatment used for allergic disorders. The present study was aimed to identify and characterize a cross-reactive allergenic protein from fungi. Allergen extracts of various fungi viz. Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Curvularia lunata, and Epicoccum purpurascens showed GST enzymatic activity ranging from 0.765 to 1.004 delta340 nm/min/microg where as activity of rGST was 1.123 delta340 nm/min/microg. Immunoblot with GST antibodies showed a band of approximately 26 kDa in all these fungal extracts. Sera of fungal allergy patients showed the presence of IgE antibodies to GST. Rabbit antibodies raised against the fungal extracts reacted with rGST confirming the presence of GST-like protein in these extract. ELISA inhibition using GST antibodies revealed inhibition with C. herbarum, A. alternata, C. lunata, A. fumigatus, and E. purpurascens demonstrating that fungal GST competes for binding to anti-GST. In summary, a GST-like protein was recognized as cross-reactive allergen in these fungal extracts.

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.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.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.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.

Fusarium solani (FS) is an important source of fungal allergen. A 45-kD major allergen of FS showed reactivity with patients' sera sensitive to many fungi.To purify and characterize a 45-kD common allergenic protein from FS, which may be useful for the diagnosis of and therapy for fungal allergy.FS culture filtrate extract was seperated on SDS-PAGE; 45-kD protein was electroeluted and purified on C18 column using reverse-phase high-pressure liquid chromatography (rpHPLC). The purified protein was functionally and biochemically characterized by in vitro and in vivo methods.The 45-kD protein showed a single peak on rpHPLC. The N-terminal amino acid sequence of this protein did not show homology to enolase or known fungal proteins. It showed cross-reactivity with Epicoccum nigrum, Curvularia lunata, Cladosporium herbarum and Alternaria alternata by ELISA and ELISA inhibition using rabbit antibodies raised against these fungi. IgE ELISA inhibition with patients' sera positive to different fungi demonstrated allergenic cross-reactivity of the 45-kD protein with other fungal extracts. This 45-kD protein released a significant amount of histamine in FS-allergenic patients.A cross-reactive 45-kD allergenic/antigenic protein was purified to homogeneity and characterized. It has prospects for use in allergen therapy.

Curvularia lunata is an important inhalant allergen. The present study was undertaken to investigate the shared IgG- and IgE-binding components among seven Curvularia species prevalent in the aerospora.Seven different Curvularia species were grown in a semisynthetic medium for 13 days. The extracts were analyzed by SDS-PAGE, immunoblot and ELISA/immunoblot inhibition using sera from C. lunata-positive patients and anti-C. lunata rabbit serum.Different Curvularia species showed 11-19 protein bands on SDS-PAGE. Proteins of 12, 20, 31, 45, 53, 78 and 97 kD were present in all the species. Eight out of 98 nasobronchial patients exhibited positive skin tests to C. lunata and to at least five Curvularia species. ELISA using these sera showed IgE binding with Curvularia species. Immunoblot using pooled anti-C. lunata sera from patients showed 5-12 allergenic proteins. Proteins of 12, 31, 45, 53 and 78 kD showed IgE binding in Curvularia species. Antibodies against C. lunata detected 6-14 antigenic proteins on immunoblot. Proteins of 31, 45 and 53 kD showed IgG binding in all the species. Proteins of 31 and 53 kD showed complete IgE/IgG binding inhibition. IgE/IgG ELISA inhibition showed dose-dependent inhibition in Curvularia species. C. lunata extract required 0.17 and 0.11 microg of protein for 50% IgE and IgG inhibition, respectively. C. clavata and C. pallescens required 10 times more protein to exhibit the same inhibition and other species required similar protein levels as those required by C. lunata.A high degree of cross-reactivity was observed between C. lunata and the six other Curvularia species tested. C. lunata and C. senegalensis shared maximum allergenic and antigenic components.

Background : Tropical countries experience wide variations in daytime temperature and relative humidity. This affects the quality of the source material used for allergen extracts. The present study was undertaken to standardize the processing and preservation conditions of Imperata cylindrica grass pollen. Methods : I. cylindrica (Ic) inflorescence were freeze-dried, pollens sieved out and stored at −70 °C (IcA). Alternatively, the inflorescence were dried at room temperature and then at 37 °C, pollens sieved out and stored at 4 °C (IcB). The extracts prepared in PBS were analyzed in vivo by skin tests and in vitro by immunochemical methods. Results : Reduced SDS-PAGE revealed 37 protein bands in IcA extract and 23 in IcB extract. IgE immunoblot with a pool of sera from Ic hypersensitive patients showed 30 allergenic bands in IcA and 14 in IcB. Immunoblot using anti-Ic rabbit sera revealed 33 antigenic bands in IcA and 22 in IcB. In both blots, the IcA extract exhibited sharp bands and the IcB extract exhibited diffuse bands. ELISA, ELISA inhibition and skin test procedures showed that IcA extracts had a higher potency than IcB extracts. Conclusions : Extracts prepared from −70 °C processed and preserved pollens (IcA) are allergenically more potent and contain a greater number of major and minor allergens than IcB extracts.

Background: Alternaria is one of the most important fungi associated with allergic disease, whereas Aspergillus fumigatus is involved in a broad spectrum of pulmonary diseases. Currently, fungal extracts used for diagnosis in the United States are unstandardized, and their allergenic content cannot be compared directly. Objective: The goal of this study was to compare the variability of major allergen levels among US allergenic products derived from fungi: specifically, Alt a 1 levels in Alternaria alternata extracts, and Asp f 1 levels in A fumigatus extracts. Methods: A novel 2-site monoclonal antibody ELISA was used for measuring Alt a 1 using recombinant Alt a 1 as a standard. Asp f 1 was also measured by ELISA. Allergenic products produced by 8 US manufacturers over a 2-year period were compared, as were multiple lots produced by a single company. Results: Alt a 1 levels in Alternaria extracts from 8 companies produced in 1998 and 1999 ranged from less than 0.01 to 6.09 μg/mL (mean 1.4 ± 1.6 μg/mL, n=15). In general, Alt a 1 levels were consistent within and between companies (1.4 ± 1.1 μg/mL, n=27), with 21 of 32 (66%) of all extracts tested containing 0.7 to 2 μg/mL Alt a 1. Aspergillus extracts showed much greater variability in Asp f 1 levels, with extracts from 8 companies containing from less than 0.1 to 64 μg/mL Asp f 1 (mean 16.3 ± 23.9 μg/mL, n=15). Overall variability was greater for Aspergillus products within and between manufacturers (22 ± 22 μg/mL Asp f 1, n=20). Conclusions: ELISA-based assays for specific allergens showed greater consistency among allergenic products derived from Alternaria than from Aspergillus. These assays should facilitate improved quality control and standardization of fungal allergen extracts and lead to the development of more consistent products for clinical use. (J Allergy Clin Immunol 2001;107:641-6.)

Background: The Epicoccum nigrum (EN) extract used in allergy disorders exhibits batch-to-batch variations in protein composition and allergenic potency. In this study, the allergens of EN grown in different media were investigated. Methods: EN was grown in five different nutrient media as stationary cultures at 25°C for 5–23 days. The growth pattern was characterized by measuring dry weight, and protein and carbohydrate content. The antigenic and allergenic content of EN extract was evaluated with EN-positive patients' sera and antibodies raised in rabbit. Results: The growth of EN in Czapeck Dox medium yielded insufficient material, while Sabouraud's broth with yeast extract (SBY) gave maximum spore-mycelial mass and protein content. Potato dextrose broth (PDB) and potato dextrose agar (PDA) showed higher dry weight and protein in 7–9-day cultures. SDS–PAGE resolved 26, 22, and 21 protein bands in EN extracts from cultures of day-13 SBY, day-7 PDB, and day-9 PDA, respectively. IgE/IgG immunoblots showed more allergenic ( 25)/antigenic ( 25) bands in EN cultured in SBY than in the others. Specific IgE ELISA and intradermal tests showed EN extract from day-13 culture in SBY to be the most potent. Conclusions: The day-13 culture of EN in SBY was the most potent and may be selected for preparing EN extracts for diagnosis of allergy and future studies.

Background: Despite enormous efforts toward the standardization of fungal extracts, only a few extracts have been characterized that are relevant for the diagnosis and immunotherapy of allergy-associated disorders. Objective: The goal of this study was to determine the optimum growth condition of Curvularia lunata , an important fungal allergen for quality raw material, and to analyze the C lunata extract for IgE- and IgG–binding proteins. Methods: C lunata was grown in synthetic (Czapeck Dox medium), semisynthetic (Sabouraud's broth [SB]), and natural media (potato dextrose [PD]) for different periods of time. The extracts were probed for allergenic and antigenic activity, with pooled patient sera and polyclonal antibodies raised against C lunata by means of ELISAs, immunoblots (in vitro), and intradermal tests (in vivo). Results: The growth of C lunata was better in semisynthetic media (ie, SB) compared with other types of media. Dry weight and protein content was maximum in the 7-day culture of SB. ELISA with pooled sera from C lunata –sensitive patients exhibited that cultures grown in SB for 11 to 13 days and PD plus 1.0% agar for 5 days were the most potent. Intradermal tests with 11- to 13-day SB culture extract showed maximum skin reactivity in allergy patients. Immunoblots with patient sera showed 10 to 14 IgE-binding proteins in 5- to 15-day SB cultures. Analysis of 5- to 15-day SB extract with rabbit sera showed 10 to 16 IgG-binding proteins. Conclusions: The extract from 11- to 13-day SB cultures were most biologically potent (intradermal tests) and showed high antigenic and allergenic reactivity (ELISA and immunoblot). The addition of yeast extract did not affect the dry weight and protein content of the C lunata extract. Furthermore, addition of agar in PD medium increases the dry weight and protein content of the fungal mat. Synthetic media was not suitable for mass cultivation of C lunata . (J Allergy Clin Immunol 1999;104:857-62.)

Background: Previous studies have established the role of Imperata cylindrica (Ic) pollen in type I allergic disorders. However, no systematic information is available on the allergen composition of Ic pollen extract. Objectives: To characterize the IgE–binding proteins of Ic pollen extract and to detect the presence of grass group 1, 4 and 5 allergen homologues, if any. Methods: Pollen extract of Ic was analyzed by in vivo and in vitro procedures such as intradermal tests (ID), enzyme–linked immunosorbent assay (ELISA), ELISA–inhibition, thin–layer isoelectric focusing (TLIEF), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting. Dot blot assay was carried out to check the presence of well–known group 1, 4, and 5 allergen homologues in Ic pollen extract. Results: Out of 303 respiratory allergies patients skin–tested, 27 showed sensitivity to Ic pollen extract. Specific IgE levels were elevated in all 15 serum samples tested. The extract prepared for this study was found to be highly potent since it required only 400 ng of homologous proteins for 50% inhibition of binding in ELISA inhibition assays. TLIEF of Ic pollen extract showed 44 silver–stained bands (pI 3.5–7.0) while SDS–PAGE resolved it into 24 Coomassie–Brilliant–Blue–stained bands (MW 100–10 kD). Immunoblotting with individual patient sera recognized 7 major IgE–binding bands (MW 85, 62, 57, 43, 40, 28 and 16 kD) in Ic pollen extract. A panel of monoclonal antibodies, specific to group 1, 4 and 5 allergens from Phleum pratense pollen extract identified group 5 and group 4 homologues in Ic pollen extract. Conclusion: Ic pollen extract was characterized for the protein profile by TLIEF and SDS–PAGE. IgE reactivity was determined by ELISA and immunoblot. Monoclonal antibodies to group 5 and group 4 allergens reacted weakly showing that this pollen contains group 5 and group 4 homologous allergens.

Fusarium equiseti is one of the most important species in the class Deuteromycetes (Fungi Imperfecti). For proper diagnosis and immunotherapy, isolation and characterization of allergens of F. equiseti are necessary. In the present study, culture filtrate (CF) extract of F. equiseti was resolved into 35-37 bands on isoelectric focusing pi (3-9) and SDS-PAGE (mol. wt. 10-100 kDa). Most of them were glycoproteins, as identified by PAS staining. F. equiseti CF revealed 15 allergenic proteins on immunoblot with an allergic serum pool. It was fractionated into nine fractions (I-IX) on a Superose-12 column by FPLC. Fraction IV (65 kDa) and fraction VI (25 kDa) were found to be highly allergenic by IgE ELISA. A 65-kDa protein was observed as a major allergen because it was recognized by most of the patient sera on immunoblot. After elution from SDS-PAGE gel, it gave two bands of pi 7.4 and 6.0. Inhibition in IgE-binding components of F. equiseti CF with CF extracts of F. soiani and F. moniliforme by immunoprint inhibition assay indicated the allergenicity shared between the extracts of Fusarium species. Data suggested that the 65-kDa is the major allergen in the Fusarium species and can he used for the treatment of allergic patients.

In vitro testing is useful for detecting pollen and fungal-allergen sensitivities in nasobronchial-allergy patients. However, allergen-coated discs or solid-phases supplied in kits do not contain all the relevant extracts. The present study was aimed to develop a safe and credible procedure of allergen coating on paper discs for allergy diagnosis by ELISA. Paper discs were coated with indigenous allergens using poly(vinyl alcohol) and glutaraldehyde. ELISAs were carried out to standardize the allergen-coated discs using skin-test-positive-patients' sera. The cut-off value (A) for ELISA was worked out by analysing normal human sera (NHS) and is based on the mean+/-2 S.D. for the NHS value. The allergen-coated discs, i.e. Centre for Biochemical Technology (CBT) discs, demonstrated excellent comparison with plate-binding method and Pharmacia discs [Prosopis juliflora (mesquite), critical value 0.879 at 5% probability+/-0.599, n=11, is significant]. Inter- and Intra-assay coefficients of variations ranged from 2.5-14.05% to 4. 8-11.5% with different allergens. Results of skin tests and ELISA with CBT discs showed 60-70% correlation.