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Notes: Background The group 7 mite allergens react with IgE in 50% of sera from allergic patients.Objective To determine the molecular and antigenic characteristics and heterogeneity of Der f 7 in mite extracts.Methods Monoclonal antibodies (MoAbs) produced from mice immunized with recombinant Der f 7 were examined for crossreactivity to Der p 7 and then used for immunoblotting of 1 and 2-D gel electrophoresis. Deglycosylation was studied with N-glycosidase-F and N-terminal sequencing by Edman degradation. The epitopes of the monoclonal antibodies were compared by cross-inhibitory immunoassays.Results Immunoblotting of D. farinae extracts with all the anti Der f 7 MoAbs showed major reactivities at 31, 30 and 25 kDa. The strongest immunostaining was at 25 kDa which contrasted with Der p 7 where the 31 and 30 kDa bands were strongest. The relative strength of staining however varied between extracts. The 31 and 30 kDa components were glycosylation products of the 25 kDa form which had the N-terminal sequence predicted from cDNA analysis. Two MoAbs stained an 18 kDa band consistent with a degradation product. The 2-D gels showed that different components with pls from 5.6–6.4. Both species-specific and Der p 7 crossreactive MoAbs were produced and a two-site ELISA assay for detecting group 7 allergen was developed with MoAbs recognizing different epitopes.Conclusions Der f 7 has been defined by its natural N-terminal sequence and MoAbs. It apparently exists as different glycosylation and degradation products in mite extracts, the relative abundance of which differs with different preparations. A two-site ELISA to measure the allergen was developed.

Notes: Background The recently characterized group 7 house dust mite allergens give positive skin-test reactions in 53% of allergie patients.Objective This study was performed to compare the IgE bindinig activity of natural and recombinant Der p 7, to measure the binding in allergic sera in comparison to major allergen Der p 2 and characterize the response by competitive inhibition with monoclonal antibodies.Methods IgE anti Der p 2 and Der p 7 antibodies against the recombinant allergens and monoclonal binding activities were measured by a solid phase radioimmune assay. Reslts A competitive binding assay showed that rDer p 7 inhibited 91% of IgE-binding to natural Der p 7 in 2 sera and 73% in a further two. The IgE binding of rDer p 2 and Der p 7 from 41 sera was then compared. Of the sera 88% and 46% respectively showed positive binding. All of the 19 sera which bound Der p 7 also bound Der p 2 but 11 (58%) had bound IgE to Der p 7 as high or higher than the binding to Der p 2. These sera were mostly high responders to both allergens. A panel of six monoclonal antibodies produced against either rDer p 7 or rDer f 7 was used for epitope analysis. All of these reaeted with eaeh allergen by enzyme linked immunosorbent assay (ELISA) and immunoblotting. Two patterns of cross inhibition of monoclonal antibody binding were observed and of five monoclonal antibodies tested, lour could inhibit the binding of IgE (WH9, WH22. WPS and HD19) while one (WH14) could not.Conclusions Although Der p 7 only reacts with 50% of allergic sera it often has a high IgE binding activity and may be more important than the major Der p 2 allergen in a high percentage of subjects. The combined competitive inhibition experiments show the IgE response is directed at several specilieities.

Notes: Plaque radio-immuno assay has been used to isolate an FgE-binding clone from a lambda gt11 library of Dermatophagoides pteronyssinus cDNA, The clone HD6 contained DNA encoding a 215 residue protein which contained a predicted 17 amino acid residue leader sequence, no cysteines and a single W-glycosylation site. The 198 residue mature protein would have a predicted MW of 22 177 D. No homologues were found in searches of the data banks. Sera from 14/38 allergic children reacted strongly with the polypeptide produced by the clone (37%). Skin tests showed reactivity in 16/30 (53%) allergic patients and 0/10 of controls. Affinity purification of rabbit antibodies with the clone showed that antibodies to the polypeptide had specificities to multiple products in mite extracts corresponding to components of Mr 29, 27 and 24 K by Western blotting. Absorption studies of IgE in allergic serum indicated further entities at 13 and 11.5 kD, It is proposed to name this allergen Der p VII.

Notes: In an earlier study we showed that Bermuda grass (Cynodon dactylon) pollen contains at least 12 IgE-binding proteins that can be analysed by immunoblot technique. One of the active components (BG-60) proved to be a basic protein of glycoprotein nature. It contained about 28% carbohydrate as determined from the dry weight and consisted of four molecules. One of the components was purified from the pollen extract by a combination of ammonium sulphate precipitation, ion-exchange chromatography on carboxymethyl-TSK, gel filtration on Ultrogel AcA 44 and chromatofocusing. Its molecular weight was approximately 60 kD by SDS PAGE and 34 kD by gel filtration chromalography. The isoelectric point of the antigen was about 9.7. The homogeneity of the antigen BG-60a was assessed by one single are of immunoprecipitation both in immanodiffusion and crossed immunoelectrophoresis and by one single band after SDS-PAGE. Its allergenicity was demonstrated by direct intradermal skin test on allergic patients and by examining IgE-binding reactivity with allergic patients' serum.

Notes: A panel of 16 monoclonal antibodies (MoAbs) directed against Bermuda grass (Cynodon dactylon) pollen (BGP) were generated for identification and purification of the major allergenic components of the eliciting antigen (Ag). Radioimmunoprecipitation (RIP) analysis revealed that there were at least eight antigenic components with molecular weights (MW) ranging from 12 kilodalton (12 kDa) to 200 kDa. Each of these components has distinct biochemical characteristics based on sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing(IEF). Among them, Cyn d Bd67K and Cyn d Bd58K were basic proteins, Cyn d Bd35K consisted of at least four isomeric components with isoelectric points ranging from 6.2 to 7.2. The other antigens Cyn d Bd68K, 48K, 38K, Cyn d Bd200K, Cyn d Bd46K, Cyn d Bd25K and Cyn d Bd12K) were all acidic proteins. The IgE binding capacity of all these antigens was determined with sera from 11 BGP-allergics by using a modified radioallergosorbent test. All but one of the antigens (Cyn d Bd200K) were found to react with human IgE from sera of BGP-allergic patients. Among those human IgE-binding molecules, Cyn d Bd35K. reacted with allergic sera most frequently (10 of 11), followed by Cyn d Bd58K (8 of 11) and Cyn d Bd46K (7 of 11) respectively. Our results suggest that Cyn d Bd35K, Cyn d Bd58K, and Cyn d Bd46K are major allergens of BGP, and the MoAbs we obtained should be valuable tools for further purificaiton of these allergens.

Notes: Background:  We have identified previously that Penicillium citrinum is the most prevalent Penicillium species in the Taipei area. It is important to delineate the whole spectrum of allergenic components of this prevalent airborne fungus. The purpose of this study was to identify novel P. citrinum allergens through molecular cloning of allergen genes using a cDNA library of P. citrinum and sera from patients with bronchial asthma.Methods:  A lambda-Uni-ZAP XR-based cDNA library of P. citrinum was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and heterologously expressed in Escherichia coli. The frequency of IgE-binding to the expressed protein and the IgE reactivity to allergen subunits were analyzed by immunoblotting.Results:  An IgE-reactive cDNA clone (clone B) was isolated by plaque immunoassay. The cDNA insert is 876-bp long and encodes a 228-amino acid polypeptide with a calculated molecular mass of 25 035 Da. Protein database search with the deduced clone B sequence revealed that 121 (53%) and 82 (36%) of the 228 amino acids were identical to those of the elongation factor 1-beta (EF-1β) proteins from the yeast Saccharomyces cerevisiae and the parasite Echinococcus granulosus, respectively. His-tagged recombinant clone B proteins were constructed and expressed in E. coli. Seven (8%) of the 92 serum samples from patients with bronchial asthma showed IgE-binding to the recombinant clone B protein. Among these seven positive sera, five demonstrated IgE-binding to the C-terminal fragment (aa 119–228) while the other two sera showed IgE reactivity to the N-terminal fragment (aa 1–118) of this newly identified EF-1βPenicillium allergen.Conclusions:  A novel P. citrinum allergen (Pen c 24) was identified and characterized in the present study. Results obtained provide more information about allergens of prevalent airborne fungi and a basis to understand more about the IgE responses in human atopic disorders and in parasitic infections.

Notes: Background:  Dermatophagoides pteronyssinus (Dp) and D. farinae (Df) mites are the most important source of indoor aeroallergens. Most Dp mite allergens identified to date have relatively low molecular weights (MWs). Identification of high-MW mite allergens is a crucial step in characterizing the complete spectrum of mite allergens and to provide appropriate tools for diagnostic and therapeutic application.Methods:  The full-length Der p 11 cDNA clone was isolated using cDNA library immunoscreening, the 5′–3′ rapid amplification of cDNA ends (RACE) system and polymerase chain reactions (PCR). The whole cDNA insert and its PCR-derived DNA fragments (p1 to p4) were generated and expressed in the Escherichia coli expression system. The allergenicity of the recombinant protein and its peptide fragments was examined by IgE immunodot assays. The IgE-binding reactivity of rDer p 11 was analyzed in the serum of 50 asthmatic children with positive reactivity to Dp mite extract. Its recombinant peptide fragments were also examined by immunodot assays in 30 mite-allergic children.Results:  Der p 11 cDNA consists of a 2625-bp open reading frame encoding a 103-kDa protein with 875 amino acids. It exhibits significant homology with the paramyosin of other invertebrates. The protein sequence alignment of this newly identified Dp mite allergen (denominated as Der p 11) revealed over 89% identity with Der f 11 and Blo t 1. Among 50 Dp-sensitive asthmatic children, rDer p 11 showed positive IgE-binding reactivity to 39 patients (78%). Using immunodot assays, multiple human IgE-binding activities were demonstrated in all four fragments of Der p 11. Using immunoblot assays, the dominant IgG-binding epitope for monoclonal antibody (mAb642) was located in fragment p3 only. In immunoblot assays, cross-inhibition between rDer p 11 and rDer f 11 was up to 73–80% at concentrations of 100 μg/ml.Conclusions:  This study confirms that the newly identified recombinant Der p 11 is a novel and important high-MW Dp mite allergen for asthmatic children. Our data also indicates that human IgE-binding major epitopes are scattered over the entire molecule of Der p 11.

Notes: Background: Candida albicans has been implicated in human allergic disorders. However, many of its immunoglobulin E (IgE)-reacting components have not yet been identified. The purpose of the present study is to characterize a novel 29 kDa IgE-binding protein from C. albicans.Methods: The 29 kDa protein was partially purified and its tryptic digests subjected to mass spectrometric analysis. The cDNA encoding this protein was isolated and heterologously expressed in Escherichia coli. Monoclonal antibodies (MoAbs) were raised against the 29 kDa protein purified from C. abicans extracts.Results: We isolated a 29 kDa IgE-reacting component from C. albicans. The protein was digested on-gel with trypsin and the masses of the resulting fragments were determined in a MALDI-TOF mass spectrometer. The data were searched against protein sequences deduced from the C. albicans genome. An open reading frame that possibly encodes the 29 kDa IgE-reacting component was identified. The cDNA corresponding to the open reading frame was isolated. It encodes a 236 residues protein that has 62% sequence identity to that of a hypothetical protein (YDR533c) from Saccharomyces cerevisiae. Conserved domain search suggests that the encoded protein belongs to the ThiJ/PfpI family. The cDNA isolated was inserted into a pQE-30 vector for protein expression in Escherichia coli. The recombinant protein can react with IgE antibodies in sera from asthmatic patients and two MoAbs that were generated against the purified native 29 kDa protein from C. albicans.Conclusions: We identified and cloned a novel 29 kDa IgE-reacting component (Cand a 3) from C. albicans. The recombinant proteins produced from this clone and the MoAbs prepared may be useful in the standardization of diagnostic extracts. They are also instrumental in elucidating the role of C. albicans in clinical allergy.

Notes: Background: We have suggested previously that the 32 and 34 kDa major allergens of Penicillium chrysogenum (also known as P. notatum) are the vacuolar (Pen ch 18) and the alkaline (Pen ch 13) serine proteases, respectively, of P. chrysogenum. The purpose of this study is to characterize the 32 kDa allergen of P. chrysogenum and its immunoglobulin E (IgE)cross-reactivity with Pen ch 13 allergen.Methods: The full-length cDNA of Pen ch 18 was isolated by reverse transcriptase-polymerase chain reaction and the 5′-rapid amplification cDNA end reaction. Recombinant Pen ch 18 was expressed as his-tagged proteins in Escherichia coli. Its reactivity with IgE and monoclonal antibodies against fungal serine protease allergens was analyzed by immunoblotting. The IgE cross-reactivity between Pen ch 18 and Pen ch 13 was analyzed by immunoblot inhibition. Overlapping recombinant fragments and synthetic peptides were used to map the B cell epitopes on Pen ch 18.Results: In this study, we isolated a 1857 bp cDNA fragment containing an open reading frame of 494 amino acids that encodes the preproenzyme of Pen ch 18. Similar to other vacuolar serine proteases, this precursor appears to undergo N- and possibly C-terminal cleavage upon maturation. The his-tagged recombinant Pen ch 18 containing the putative sequence of the mature protein reacted with IgE antibodies in serum samples from asthmatic patients. In addition, IgE-binding to the 32 kDa major allergen of P. chrysogenum was inhibited when a positive serum sample was absorbed with recombinant Pen ch 18 before immunoblotting. Both inhibition and almost no inhibition of IgE-binding to the 32 kDa major allergen of Pen ch 18 were detected when eight positive serum samples were preabsorbed individually with purified Pen ch 13 before immunoblotting. The major IgE binding region was located in a fragment (PN1) encompassing the N-terminal 102 amino acid residues of the recombinant Pen ch 18. A dominant linear IgE epitope was further mapped within residues 73–95 (peptide PN1-e) of the N-terminally processed allergen. Monoclonal antibody FUM20 that reacts with Pen ch 18 but not with Pen ch 13 binds a synthetic peptide with sequence encompassing the N-terminal 23 residues of the recombinant Pen ch 18. Monoclonal antibody PCM39 that reacts with both Pen ch 13 and Pen ch 18 recognizes a peptide containing residues 132–154 of the allergen.Conclusions: Our results confirm that the Pen ch 18 allergen is a vacuolar serine protease of P. chrysogenum that matures through N- and possibly C-terminal processing. The finding that there are cross-reactive and allergen-specific IgE epitopes for Pen ch 18 and Pen ch 13 suggests that both major allergens should be included in clinically diagnostic P. chrysogenum extracts.