Win-g Lin

The 40‐kilodalton allergen of Candida albicans is an alcohol dehydrogenase: molecular cloning and immunological analysis using monoclonal antibodies

To characterize the 40‐kilodalton (kD) major allergen of Candida albicans (C. albicans), six monoclonal antibodies (MoAbs) against this allergen were generated. In SDS‐polyacrylamide gel electrophoresis and immunoblot analysis, these MoAbs showed four different reaction patterns to antigens of six different Candida species. With the exception of one MoAb, other MoAbs were resistant to periodate treatment indicating non‐carbohydrate epitopes were probably being recognized by these MoAbs. These MoAbs were used in the molecular cloning and immunological analysis of the gene coding for the 40‐kD allergen. Nucleotide sequence determination of the two λgtl 1 cDNA clones obtained showed that the 40‐kD allergen is an alcohol dehydrogenase (ADH) which shares a 70% amino acid sequence homology with the ADH isozyme I of Saccharomyces cerevisiae. This finding was confirmed by positive immunological response of the lysates of the clones obtained and a preparation of ADH of Saccharomyces cerevisiae to various MoAbs and to IgE antibodies in sera of allergic patients.

Alkaline Serine Proteinase: A Major Allergen of Aspergillus oryzae and Its Cross-Reactivity with Penicillium citrinum

Background:Aspergillus species are common indoor airborne fungi and have been considered as causative agents of human allergic disorders. However, allergens of different Aspergillus species have not been effectively characterized. The object of this study was to identify and characterize IgE-binding components of Aspergillus oryzae. Methods. Allergens of A. oryzae were identified by immunoblot analysis using sera from asthmatic patients. The N-terminal amino acid sequences of allergens thus identified were determined by Edman degradation. The antigenic and the allergenic cross-reactivities between allergens of different fungi were analyzed by immunoblotting and immunoblot inhibition analysis, respectively, using a monoclonal antibody (MoAb) 55A against the 33-kD major allergen of Penicillium citrinum and a mixture of IgE-containing asthmatic serum samples. Results: Thirteen components of A. oryzae ranging in apparent molecular weight from 16 to 42 kD react with IgE antibodies. A 34-kD component that showed intense IgE-binding reactivity and was detectable in the highest frequency in our asthmatic serum samples tested was considered a major allergen of A. oryzae. The 34-kD component also reacted with MoAb 55A. Results from immunoblot inhibition studies also demonstrated the IgE cross-reactivity between the 34-kD major allergens of A. oryzae and P. citrinum. In addition, the sequence of the N-terminal 18 amino acid residues of the 34-kD major allergen of A. oryzae was found to be identical to that of the alkaline serine proteinase from the same Aspergillus species. Conclusion: The 34-kD major allergen of A. oryzae is an alkaline serine proteinase. There is IgE cross-reactivity between the major serine proteinase allergens of A. oryzae and P. citrinum.

Identification of vacuolar serine proteinase as a major allergen of Aspergillus fumigatus by immunoblotting and N-terminal amino acid sequence analysis.

Aspergillus species are common airborne fungi that have been identified as causative agents of extrinsic bronchial asthma. More than 10 allergens from A. fumigatus have been recently characterized by cDNA cloning.

Alkaline Serine Proteinase Is a Major Allergen of Aspergillus flavus, a Prevalent Airborne Aspergillus Species in the Taipei Area

Background: Aspergillus species are prevalent indoor airborne fungi and have been identified to be a causative agent of human allergic disorders. In the present study, we identified, purified and characterized the allergen(s) from Aspergillus flavus, a predominant airborne Aspergillus species in the Taipei area. Methods: The IgE–binding components of A. flavus were identified by SDS–PAGE immunoblotting with sera from asthmatic patients. The N–terminal amino acid sequences of the major allergens were determined by Edman degradation. The allergenic cross–reactivity among allergens from different fungi was analyzed by immunoblot inhibition using sera from asthmatic patients. The detected major allergen was purified from the culture medium. It was further characterized in terms of its N–terminal amino acid sequence, its IgE–binding activity and its enzymatic activity. Results: The results of the immunoblot analysis indicate that a 34–kD component that has high IgE–binding (63%) frequency is a major allergen of A. flavus. The N–terminus of this 34–kD major allergen (GLTTQKSAP) has high sequence identity with that of the 34–kD alkaline serine proteinase major allergen of A. oryzae. Results from immunoblot inhibition studies indicate that IgE cross–reactivity occurs among the 34–kD major allergens of A. flavus, A. fumigatus and Penicillium citrinum. The 34–kD major allergen of A. flavus was purified from the culture medium by ammonium sulfate precipitation and DEAE ion exchange chromatography. The N–terminal amino acid sequence of the purified allergen (Asp fl 13) is identical to that determined previously for the 34–kD major allergen in the crude extract of A. flavus. The IgE immunoblot reactivity to the 34–kD major allergen in the crude extract can be dose–dependently inhibited by the purified Asp fl 13. The degree of IgE binding to the 34–kD major allergen in the crude extract correlates with that of the purified Asp fl 13 in sera of 8 asthmatic patients. The purified Asp fl 13 has proteolytic activity with casein as substrate at pH 8.0. This enzymatic activity can be inhibited by either phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Conclusion: Our results suggest that the 34–kD alkaline serine proteinase is a major allergen of A. flavus. There was IgE cross–reactivity among allergens of A. flavus, A. fumigatus and P. citrinum.

Allergenic components in three different species of Penicillium: crossreactivity among major allergens

Background Penicillium species have been considered as important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous airborne fungal species.

Characterization of the 33‐kilodalton major allergen of Penicillium citrinum by using MoAbs and N‐terminal amino acid sequencing

Background The 33 kD component has been identified as a major allergen ofPenicillium citrinum, the most prevalent Penicillium species in the Taipei area of Taiwan.

Production and characterization of monoclonal antibodies to serine proteinase allergens in Penicillium and Aspergillus species

Alkaline and/or vacuolar serine proteinases are major allergens in prevalent airborne Penicillium and Aspergillus species.

Molecular cloning and expression of a Penicillium citrinum allergen with sequence homology and antigenic crossreactivity to a hsp 70 human heat shock protein

Background Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. However, detailed studies on allergens of this ubiquitous Penicillium species are still lacking.

Characterization of a monoclonal antibody (P40) against the 68 kD major allergen of Penicillium notatum

A monoclonal antibody (MoAb P40) against the 68 kD major allergen of penicillium notatum (P. notatum) was obtained by immunizing the mouse with a crude extract of P. notatum. Analysed by two‐dimensional gel electrophoresis and immunoblotting, P40 reacted with two different isoforms of the 68 kD component of P. notatum with pIs of 5.4 and 5.5. In addition to P. notatum, P40 showed positive ELISA activity to Aspergillus fumigatus (A. fumigatus) but not to components of six other fungi including Alternaria porri, Cladosporium cladosporoides, Aureobasidium pullulans, Fusarium solani, Rhizopus arrhizus and Candida albicans. Analysed by ELISA, MoAb P40 also showed positive activity to two (P. frequentans and P. roseopurpureum) of the 10 other Penicillium species and two (A. terreus and A. flavus) of the four other Aspergillus species tested. SDS‐PAGE and immunoblotting studies demonstrated P40 positive reactivity to components with MW of about 67 kD in all these Penicillium and Aspergillus species with positive ELISA activity to P40. Furthermore, immunoblotting activity of MoAb P40 to the 67 kD component of A. niger was also observed. The epitope of the 68 kD allergen of P. notatum recognized by MoAb P40 was resistant to treatment of periodate oxidation with concentration of NaIO4 up to 20 mm. This MoAb may thus be useful in the characterization and purification of the 68 kD allergen from crude extracts, and in the molecular cloning of allergen genes.

113 Allergenic components in three different species of pencillium: Cross-reactivity among major allergens

BACKGROUND Penicillium species have been considered as important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous airborne fungal species. OBJECTIVE This study compares the allergenic profiles and allergenic crossreactivity among allergens of three prevalent airborne Penicillium species. METHODS IgE-binding Penicillium components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-immunoblotting using sera from 67 asthmatic patients. The presence of allergenic crossreactivity was analysed by immunoblot inhibition. RESULTS Among the 67 serum samples tested, 15, 14 and 11 samples showed IgE reactivity to components of P. citrinum, P. notatum and P. brevicompactum, respectively. All 15 P. citrinum-positive serum samples showed IgE-binding to a 33 kDa extract component of this species. Thirteen (93%) of the 14 P. notatum-positive serum samples and 10 (91%) of the 11 P. brevicompactum-positive sera also showed IgE reactivity to components with a molecular weight of about 33 kDa in individual Penicillium species. All of the 10 P. brevicompactum 33 kDa component-positive serum samples showed IgE reactivity to the 33 kDa components of the other two Penicillium species tested. Dose-dependent inhibition of IgE-binding to these major allergens was observed when the positive serum sample was absorbed with different amounts of individual allergenic extract as well as with different amounts of extracts prepared from the other two Penicillium species. CONCLUSION Although different allergenic profiles were observed in the three different Penicillium species tested, results showed that there was an IgE crossreactivity among the 33 kDa group major allergens of P. citrinum, P. notatum and P. brevicompactum.