Jyotsna Verma

Fusarium solani: Immunochemical Characterization of Allergens

Allergenic components of the fungus Fusarium solani were isolated using (NH4)2SO4 precipitation and ion-exchange column chromatography. The allergenicity of fractions was studied by enzyme-linked immunosorbent assay and radioallergosorbent test inhibition techniques. Proteins of culture filtrate (CF), mycelium (MY), and spore (SP) extracts of F. solani were characterized by isoelectrofocusing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and IgE-specific immunoblotting. CF antigen of F. solani contained more allergenic proteins than MY and SP, visible on immunoblot analysis using allergenic serum pool. A 65-kD protein component of CF was found to be a major allergen, as it was strongly visible on immunoblots of all 15 patient sera tested. Crossed immunoelectrophoresis and enzyme-linked immunosorbent assay inhibition using rabbit antibodies raised against F. solani CF demonstrated shared antigenicity between CF, MY, and SP extracts. It was observed that F. solani is a significant allergen, and most of the allergens of MY and SP extracts were found in CF extract. Therefore, CF alone can be used in the preparation of a standard extract. However, few unique allergenic proteins were observed in MY as well as in SP extracts of F. solani. Hence, the use of combined CF, MY, and SP extracts of F. solani is recommended for diagnosis and immunotherapy.

Purification and characterization of a cross-reactive 45-kD major allergen of Fusarium solani.

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.

Immunobiochemical Characterization of Brassica campestris Pollen Allergen

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.

Immunobiochemical Characterization of Putranjiva roxburghii Pollen Extract and Cross-Reactivity with Ricinus communis

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 characterized and compared with pollen allergens of Ricinus communis (RC) belonging to family Euphorbiaceae. On isoelectric focusing, PR pollen extract resolved into 35 bands (pI 3-9), whereas SDS-PAGE separated it into 18 protein components (MW 14-100 kD). Pooled patients sera (ID +ve to PR) recognized 12 allergenic proteins in Putranjiva and five of them (MWs 92, 80, 55, 43 and 30 kD) showed immunologic reactivity to most of the sera samples tested individually by immunoblot. A number of shared allergenic proteins (MWs 92, 80, 66, 50, 43 and 14 kD) were observed between PR and RC pollen extracts on immunoblot using Putranjiva allergic serum pool. Inhibition in the binding for most of PR pollen allergenic proteins was obtained with higher concentration of RC extract than PR itself, depicting the presence of cross-reacting allergens in both. Putranjiva pollen extract was fractionated by a combination of DEAE Sephadex-A 50 and Sephadex-G 200 column chromatography. Periodate deglycosylation of western blotted PR extract and Put I fraction indicated the involvement of carbohydrate moieties in the allergenic activity. Of the two fractions from Put I (Ia and Ib), Put Ib was found to be the most allergenic protein by ELISA inhibition. Dot blot analysis with individual patients sera identified it as a major allergen of PR.

Purification and Partial Characterization ofa 67-kD Cross-React ive Allergen from Imperata cylindrica Pollen Extract

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.

Purification and characterization of Fus sI3596, a 65 kd allergen of Fusarium solani

A component ofFusarium solani (F. solani), identified as the major allergen,Fus sI3596* was purified to homogeneity from culture filtrate (CF) by means of anion-exchange column chromatography, gel filtration and FPLC. The homogeneity ofFus sI3596* was assessed by IEF, PAGE, SDS-PAGE (non-reducing), immunoblot and HPLC.Fus sI3596* was isolated as a glycoprotein of MW 65 kd and pI 3.6. The IgE ELISA-inhibition assay after periodate treatment of the fraction showed a lower IgE binding capacity suggesting involvement of carbohydrate moiety in IgE binding reactions of the allergen. Peptide fragments ofFus sI3596* obtained after CNBr and trypsin treatment were analysed by immunoblotting for their allergenicity. This study indicated that there could be at least 3 allergenic determinants in the major allergen,Fus sI3596* ofF. solani CF.