Patent Application: US-74488508-A

Abstract:
the present invention relates to the field of ige - mediated allergy , particularly occupational asthma such as bakers asthma . more specifically the invention relates to the identification of a novel wheat allergen and the use thereof in therapy and diagnosis of ige - mediated allergy . furthermore the present invention provides the use of known peptides and proteins in therapy and diagnosis . the invention also relates to methods for diagnosis and treatment of ige - mediated allergy in mammals .

Description:
the examples below illustrate the present invention with the isolation and use of the polypeptide of the invention . the examples are only illustrative and should not be considered as limiting the invention , which is defined by the scope of the appended claims . clone # 123 mentioned in the examples is profilin , known from e . g . u . s . pat . no . 7 , 214 , 786 . example 1 shows the identification and characterization of a novel wheat seed allergen , clone # 10 , belonging to the potato inhibitor family , a family of serine protease inhibitors , which together with other protease inhibitors are referred to as pathogenesis related proteins ( pr ), family pr6 . clone # 10 is the first allergen identified and described for the pr6 family . furthermore example 1 shows the expression and purification of a recombinant clone 10 - derived allergen . clone # 10 was specifically recognized by serum ige from patients with baker &# 39 ; s asthma but showed no ige reactivity when tested with sera from patients suffering from food allergy to wheat , celiac disease or grass pollen allergy . therefore the clone 10 - derived allergen together with other wheat allergens may be used to establish diagnostic tests which allow to specifically identify patients suffering from ige - mediated baker &# 39 ; s asthma and to discriminate these patients from allergic patients with food or pollen allergy . wheat seeds from triticum aestivum cv . michael were obtained from the österreichische agentur für gesundheit and ernährungssicherheit gmbh and planted in a glasshouse . immature seeds were harvested 7 , 10 , 15 , 20 , 25 , 30 and 35 days after the onset of pollination directly into liquid nitrogen and stored at − 80 ° c . until use . wheat pollen was obtained from allergon ( välinge , sweden ). rice , maize , beans and potatoes were bought at a local market . recombinant phl p 1 , phl p 5 , phl p 7 and phl p 12 were purchased from biomay ( vienna , austria ) and human serum albumin ( hsa ) from behring ( marburg , germany ). sera were obtained from 22 patients suffering from bakers &# 39 ; asthma . baker &# 39 ; s asthma was diagnosed on the basis of a positive case history , ige specific for wheat and rye flour by cap - feia system ( phadia , uppsala , sweden ) and included specific inhalation challenge tests for confirmation of a clinically relevant sensitization ( 1 ). demographic , clinical and serological data of these patients are summarized in table i . in addition , serum from a non - allergic individual , sera from 4 patients suffering from food allergy to wheat and 4 grass pollen allergic patients without baker &# 39 ; s asthma but serum ige reactivity to wheat and rye flour were included in the experiments ( table ii ). sera from grass pollen allergic patients had been analyzed for total serum ige levels and ige specific timothy grass pollen by cap - feia system ( phadia ) and patients with food allergy to wheat have been characterized as described previously ( 2 ). the specificity of the clone 10 - derived allergen for baker &# 39 ; s asthma was confirmed by testing additional 20 sera from celiac disease patients , 119 food allergic patients , 23 sera from grass pollen allergic patients and 25 baker &# 39 ; s asthma patients by chip analysis ( constantin et al , unpublished ). specific rabbit antibodies against the clone 10 - derived allergen were raised by immunization of a rabbit in monthly intervals with purified clone 10 - derived allergen ( 200 μg per injection ) using once freud &# 39 ; s complete adjuvant and twice ifa ( charles river , kisslegg , germany ). pre - immune serum was obtained from the rabbit before immunization . for control purposes , a rabbit immune serum specific for a house dust mite allergen and rabbit antiserum specific for wheat profilin were used . total rna was extracted according to yeh ( 3 ) from wheat seeds , harvested twenty five days after the onset of pollination , and stored at − 80 ° c . then the rna pellet was dissolved in guanidinium isothiocyanate buffer ( 4m guanidinium isothiocyanate , 0 . 83 % v / v 3m sodium acetate , ph 6 , 11 mm β - mercaptoethanol ) and purified by cesium chloride density gradient ultracentrifugation ( 4 ). poly - a + rna was isolated by oligo - dt cellulose affinity chromatography ( nucleo trap mrna ; machery - nagel ) and double stranded cdna was synthesized with a cdna synthesis kit ( cdna synthesis system ; roche diagnostics , mannheim , germany ). after methylation with ecori methylase ( new england biolabs , beverly , mass . ), ecori linkers ( new england biolabs ) were added to the cdna . linkered cdna was digested with ecori ( roche diagnostics ). digested linkers were removed with a nick column ( pharmacia biotech , uppsala , sweden ) and the cdna was ligated into λgt11 arms ( stratagene , la jolla , calif ., usa ). the ligation product was packed in vitro ( gigapack iii gold cloning kit , stratagene ) resulting in a λgt11 expression cdna library with 2 . 43 × 10 6 pfu . isolation and characterization of ige - reactive clones from a wheat seed cdna library e . coli y1090 were infected with 7 × 10 5 pfu of recombinant phages and immunoscreened with serum ige of four patients (# 1 , # 2 , # 4 , # 12 ) suffering from bakers &# 39 ; asthma as described ( 5 ). fifteen ige - reactive phage clones were selected for further re - cloning and their dna was pcr - amplified using platinum pcr supermix ( invitrogen , life technologies ) with λgt11 primers and sequenced ( mwg , ebersberg , germany ). the obtained sequences were compared with sequences submitted to the genbank database at the national center for biotechnology information ( ncbi ). multiple sequence alignment was performed using the genbank database at the ncbi . for amino acid sequence identities the clustal w multiple alignment tool was used . a motif search was carried out with the prosite tool of the expasy proteomics server , for amino acid composition the protparam tool of expasy was used . solvent accessibility and secondary structure prediction was calculated using the prot software from the columbia university bioinformatics center . a phylogenetic tree was reconstructed based on the amino acid sequence of the clone 10 - derived allergen and homologous proteins using the “ multiple alignment and phylogenetic tree reconstruction ” software provided by the max planck institute for molecular genetics . the coding region of the clone 10 cdna was amplified by pcr using the following primer pair : forward 5 ′- catatgagccctgtggtgaagaagccggaggga - 3 ′ and reverse , 5 ′- gaattc ttagtgatggtgatggtgatggccgaccctggggac - 3 ′ ( mwg ). the pcr product contained ndei ( italics ), ecori ( underlined ) restriction sites and a hexahistidine tag - encoding sequence ( bold ). the pcr product was subcloned into an acceptor vector ( novagen , madison , wis .) and sequenced again ( mwg ). then the insert was cut out of the acceptor vector with ndei and ecori ( roche diagnostics ), gel - purified ( promega , madison , wis ., usa ) and subcloned into the expression plasmid pet 17b ( novagen ). the dna sequence was confirmed by sequencing both dna strands ( microsynth , balgach , ch ). the pet 17b - clone 10 construct was transformed into e . coli bl21 ( de3 ) ( stratagene ) and grown in luria broth ( 17 ) medium containing 100 mg / l ampicillin at 37 ° c . to an od ( 600 nm ) of 0 . 8 - 1 . protein expression was induced by addition of isopropyl β - d - thiogalactopyranoside ( iptg ) to a final concentration of 0 . 5 mm and growing the bacteria for additional 3 h . bacteria were harvested by centrifugation and — homogenized in 25 mm imidazole , ph 7 . 5 , 0 . 1 % ( v / v ) triton x - 100 with an ultraturrax ( ika , stauffen , germany ). dna was digested by addition of dnase i , stirred for additional 10 min at 20 ° c ., the reaction was stopped with 200 μl of 5m nacl and then centrifuged at 4 ° c . ( 6000 × g , 20 min ). the majority of the clone 10 - derived allergen was found in the insoluble fraction of the bacterial extract . clone 10 - derived allergen was purified from the inclusion body - containing pellet under denaturing conditions using ni - nta resin affinity columns according to the qiaexpressionist handbook ( qiagen , hilden , germany ). fractions containing the recombinant allergen were pooled and dialyzed against 10 mm nah 2 po 4 ph 7 . 5 . the protein concentration was determined with a micro bca protein assay kit ( pierce , rockford , ill .). clone 10 - derived allergen was dissolved in pbs at a concentration of 5 μg / ml and coated on elisa plates ( nunc maxisorb , roskilde , denkmark ). after blocking with 1 % ( w / v ) bsa in pbs , 0 . 05 % ( v / v ) tween 20 ( pbst ), plates were incubated with sera diluted 1 : 50 in pbst , 0 . 5 % ( w / v ) bsa for measurement of igg 1 , igg 2 , igg 3 and igg 4 as described ( 6 ). bound antibodies were detected by incubating first with monoclonal mouse anti - human igg subclass antibodies ( bd biosciences , franklin lakes , n . j .) diluted 1 : 1000 in pbst , 0 . 5 % ( w / v ) bsa , and then with a horseradish - peroxidase - coupled sheep anti - mouse antiserum ( ge healthcare , little chalfont , uk ) diluted 1 : 2000 in pbst , 0 . 5 % ( w / v ) bsa as previously described ( 2 ). all determinations were performed as duplicates , and results are expressed as mean values . sds - protein extracts from mature and immature wheat seeds , rice ( oryza sativa ), maize ( zea mays ), common bean ( phaseolus vulgaris ) and potato ( solanum tuberosum ) were prepared by homogenization of 3 grams of tissue in 32 ml of sample buffer ( 6 ) and subsequent boiling for 10 minutes . in order to remove insoluble particles , the extracts were centrifuged at 10 , 000 × g for 10 min at 4 ° c . and the supernatants were stored in aliquots at − 20 ° c . in addition a pbs - protein extract from wheat seeds was prepared as previously described ( 2 ). triticum aestivum pollen ( 500 mg ) were extracted at 4 ° c . over night in 5 ml pbs , 2 mm edta , 1 mm pmsf . after centrifugation for 1 h at 13 , 000 × g 4 ° c ., the protein concentration of the supernatant was determined with micro bca protein assay kit ( pierce ) and aliquots were stored at − 20 ° c . until use . equal amounts of sds - protein extracts were separated by 14 % preparative sds - polyacrylamide gels ( 7 ). a protein molecular weight marker ( rainbow marker , ge healthcare ; precision plus protein standard , biorad , herkules , calif . ; page ruler pre - stained protein ladder , fermentas , burlington , ontario ) was used as a standard . after electrophoretic separation , proteins were either stained with coomassie brilliant blue or blotted onto nitrocellulose membranes ( schleich 85 schuell , dassel , germany ) ( 8 ). membranes were blocked in buffer a ( 50 mm sodium phosphate buffer , ph 7 . 4 , 0 . 5 % w / v bsa , 0 . 5 % v / v tween - 20 , 0 . 05 % w / v nan 3 ) twice for 10 min and once for 30 min and incubated overnight at 4 ° c . with a rabbit antiserum specific for the clone 10 - derived allergen , the corresponding pre - immune serum , and for control purposes , with a rabbit antiserum specific for an unrelated antigen or buffer alone . rabbit sera were diluted 1 : 50 , 000 in buffer a . bound antibodies were detected with 1 : 2000 in buffer a diluted 125 i - labelled anti - rabbit antibodies from donkey ( ge healthcare ) for 2 h at room temperature and visualized to kodak xomat films with intensifying screens ( kodak , heidelberg , germany ) at − 70 ° c . for ige dot blot experiments , 100 ng of recombinant clone 10 - derived allergen and recombinant grass pollen allergens , phl p 1 , phl p 5 , phl p 7 and phl p 12 , as well as 3 μg of wheat pollen extract and 2 μg of mature wheat seed pbs - extract were dotted onto a nitrocellulose membrane . the nitrocellulose strips were blocked with buffer a and exposed to patients sera at a 1 : 10 dilution in buffer a over night at 4 ° c . bound ige antibodies were detected with 125 i - labelled anti - human ige antibodies ( rast ria , demeditec diagnostics , germany ) diluted 1 : 20 in buffer a over night at room temperature and visualized by autoradiography using kodak xomat films with intensifying screens ( kodak ) at − 70 ° c . laser desorption mass spectra were acquired in a linear mode with a tof compact maldi ii instrument ( kratos , manchester , uk ; pichem , research and development , graz , austria ). samples were dissolved in 10 % acetonitrile ( 0 . 1 % trifluoroacetic acid ), and α - cyano - 4 hydroxycinnamic acid ( dissolved in 60 % acetonitrile , 0 . 1 % trifluoroacetic acid ) was used as a matrix . for sample preparation a 1 / 1 mixture of protein and matrix solution was deposited onto the target and air - dried . cd measurements were performed with purified clone 10 - derived allergen ( in h 2 o ) at a protein concentration of 0 . 1 mg / ml on a jasco j - 810 spectropolarimeter ( tokyo , japan ) using a 0 . 2 cm path length rectangular quarz cuvette . far - uv cd spectra were recorded from 190 nm to 260 nm with 0 . 5 nm resolution at a scan speed of 50 nm / min and resulted from the average of three scans . results are expressed as the mean residue ellipticity ( θ ) at a given wavelength . temperature scans were performed according to a step - scan procedure , where the sample was heated from 25 ° c . to 95 ° c . with a heat rate of 2 ° c ./ min and cooled back to 25 ° c . at the same rate . every 5 ° c . continuous wavelength spectra were recorded with the specified parameters . in addition , temperature scans were recorded at 215 nm with a step resolution of 0 . 5 ° c . results are expressed as the molar mean residue ellipticity ( θ mre ) at a given wavelength . the final spectra were corrected by subtracting the corresponding baseline spectrum obtained under identical conditions . the secondary structure content of clone 10 - derived allergen was calculated using the secondary structure estimation program cdsstr ( 9 ). for the quantification of ige ab - mediated , immediate - type reactions , hurbl cell mediator release assays were performed . rbl cells ( clone rbl - 703 / 21 ) transfected with the human fcεri ( 10 ) were cultured in rpmi 1640 supplemented with 5 % fcs , 4 mm l - glutamine , and 1 mg / ml g418 sulfate . cells were harvested after incubation with trypsin / edta , washed , re - suspended in culture medium , and the cell concentration was adjusted to 2 × 10 6 cells / ml . fifty μl aliquots of the cell solution were added to the wells of a 96 - well flat - bottom microplate ( cell density / well was 1 × 10 5 cells ). human sera were diluted 1 : 10 in culture medium , added to the cells and incubated overnight at 37 ° c ., 7 % co 2 , 95 % relative humidity . medium was removed and the plates were washed 3 times with 200 μl / well of tyrode &# 39 ; s buffer + 0 . 1 % bsa . for ige cross - linking , 100 μl of clone 10 - derived allergen or rphl p 1 , ( 0 . 3 μg / ml ), diluted in tyrodes &# 39 ; s buffer containing 50 % d 2 o and 0 . 1 % ( w / v ) bsa were added to the cells . for spontaneous release , tyrodes &# 39 ; buffer without protein was added to the wells . total release was determined by addition of tyrode &# 39 ; s buffer containing 10 % triton x - 100 . after incubation at 37 ° c ., 7 % co 2 , 95 % relative humidity for 1 hour cells were harvested by centrifugation and 50 μl supernatant was transferred to a new plate , and 50 μl assay solution ( 0 . 1m citric acid or sodium citrate , ph 4 . 5 and 160 μm 4 - methyl umbelliferyl - n - acetyl - β - d - glucosaminide ) per well was added . after another one hour incubation the reaction was stopped by adding 100 μl glycine buffer ( 0 . 2m glycine , 0 . 2 % nacl , ph 10 . 7 ) to each well . fluorescence was measured at λ ex : 360 / λ em : 465 in a fluorescence microplate reader . spontaneous release was determined from control wells that had not been lysed by triton x - 100 . specific release was calculated using the formula : ( sample - spontaneous / total - spontaneous )× 100 . dry grains of wheat were cut into small pieces ( cubes of approximately 0 . 5 mm size ) using a sharp razor blade . in order to preserve the dry state of the cells , the cubes were anhydrously fixed in acrolein vapor for 5 days at room temperature . they were transferred at room temperature for 1 day to dimethoxypropane ( dmp ) for removing any residual water and embedded into lowicryl k4m resin using ascending series of dmp : ethanol and ethanol : monomeric lowicryl k4 m as intermediate stages . polymerization was performed at − 35 ° c . ultrathin sections were cut from both peripheral and central grain tissues and placed on silver grids for immunolabeling procedures . labelling for clone 10 - derived allergen was performed in a moist chamber at room temperature ( pbs buffer + 1 % ( w / v ) bsa , ph 7 . 4 , tris buffer + 1 % ( w / v ) bsa , ph 8 . 2 ) as follows : 1 . 5 % ( w / v ) bsa in pbs buffer , 15 minutes ; 2 . rabbit anti - wheat protein 10 antibodies and pre - immune antibodies , diluted 1 : 35 in pbs buffer , 2 hours ; 3 . pbs buffer , 5 minutes , tris buffer , 2 × 5 minutes ; 4 . goat anti - rabbit igg antibodies coupled to colloidal gold particles of 10 nm size ( biocell , plano , wetzlar , germany ), diluted 1 : 20 in tris buffer ; 5 . tris buffer , 1 × 5 minutes , distilled water , 2 × 5 minutes . sections were stained using uranyl acetate ( 5 minutes ) and lead citrate ( 10 seconds ). samples were analyzed in a transmission electron microscope em 410 ( fei , eindhoven , the netherlands ). isolation and characterization of a wheat cdna coding for a serine proteinase inhibitor - like allergen a wheat seed cdna expression library was screened with ige antibodies from four patients suffering from bakers &# 39 ; asthma . the open reading frame of the cdna of the ige - reactive clone 10 contained 262 nucleotides coding for a 84 amino acids polypeptide ( fig1 ). a molecular mass of 9 . 4 kda and an isoelectric point ( pi ) of 6 . 08 was calculated according to the deduced amino acid sequence for the clone 10 - derived allergen . the analysis of amino acid composition showed a high content of valin residues ( 15 . 5 %) and absence of cysteine residues . according to computer - aided secondary structure analysis the clone 10 - derived allergen consists mainly of random coils and beta - sheets and one alpha - helical domain . according to solvent accessibility calculations almost 80 % of the amino acids are solvent exposed . a search for sequence motifs revealed the presence of one potential casein kinase ii phosphorylation site ( amino acid 32 ), two n - terminal myristoylation sites ( amino acids 11 , 55 ) and a potato inhibitor i family signature ( fig1 : amino acids 25 - 36 are boxed ). a comparison of the clone 10 - derived amino acid sequence with sequences deposited in the ncbi database showed that the allergen is almost identical to a triticum aestivum subtilisin - chymotrypsin inhibitor wsci precursor ( accession no . gi | 122065237 ) and to t . aestivum wsci proteinase inhibitor ( accession no . gi | 66356278 ) and exhibits significant sequence homologies with a group of serine proteinase inhibitors occurring in plants and animals . these serine proteinase inhibitors constitute a family designated potato inhibitors i and are characterized by a typical consensus sequence pattern which is conserved in each of the proteins . the serine proteinase inhibitors belonging to the potato inhibitor i family are small proteins of 60 - 90 amino acids lacking disulphide bonds and contain only a single inhibitory site . the sequences of wheat serine proteinase inhibitors show a significant degree of sequence conservation to proteinase inhibitors of other monocotyledonic plants like barley ( hordeum vulgare ), maize ( zea mays , zea diploperennis ), gamma grass ( tripsacum dactyloides ) and rice ( oryza sativa ), dicotyledonic plants and worms ( table iii ). table iii displays the percentages of sequence identities between each of the serine proteinase inhibitors . expression , purification and physicochemical characterization of the serine proteinase inhibitor - like allergen the clone 10 - derived allergen was expressed in e . coli bl21 ( de3 ) with a c - terminal hexahistidine tag . approximately 25 mg / l liquid culture of serine proteinase inhibitor - like allergen could be purified by nickel chromatography ( fig2 a ). maldi - tof analysis of purified recombinant protein 10 resulted in a mass peak of 9970 . 8 da ( fig2 b ). the far - uv cd spectrum of purified recombinant clone 10 - derived allergen ( fig2 c ) indicates that the protein is folded and contains a considerable amount of β - sheets and a low α - helical content . the spectrum is characterized by a minimum at 204 nm and a maximum at 190 nm . secondary structure analysis using the program cdsstr with the reference dataset 7 yielded 8 % α - helix , 23 % β - sheets , 14 % β - turns and 53 % random coils . the nrmsd value of 0 . 033 demonstrated a good fit between the calculated and the experimentally derived spectra . upon heating to 95 ° c . a slight shift of the minimum of the cd spectrum ( from 204 nm to 201 nm ) was observed , indicating a partial denaturation of the protein . upon cooling to 25 ° c . the protein refolded ( fig2 c ). however , the minimum at 204 nm was lower than before heating , which suggests a rearrangement of the β - sheets . in summary , the spectra observed during the temperature scan point to a high thermal stability of the clone 10 - derived allergen . recombinant clone 10 - derived allergen is a new serine proteinase inhibitor - like allergen from wheat purified clone 10 - derived allergen was tested for ige reactivity using sera from patients suffering from bakers &# 39 ; asthma , grass pollen allergy and food allergy to wheat by dot blot analysis ( fig3 ). the recombinant clone 10 - derived allergen reacted with ige - antibodies from 3 (# 1 , # 4 , # 12 ) out of 22 patients suffering from bakers &# 39 ; asthma ( 13 . 6 %). patient # 1 who had only low ige levels specific for the major wheat allergen , alpha amylase inhibitor , showed strong ige reactivity to the serine proteinase inhibitor - like allergen . interestingly , the clone 10 - derived allergen was exclusively recognized by ige antibodies from patients with baker &# 39 ; s asthma but not by patients suffering from ige - mediated food allergy to wheat or patients suffering from grass pollen allergy . several sera from each of the three patients groups showed ige reactivity to recombinant timothy grass pollen allergens due to co - sensitization to grass pollen . the specific ige reactivity of the clone 10 - derived allergen by baker &# 39 ; s asthma patients was confirmed in a chip analysis using additional 20 sera from celiac disease patients , 119 food allergic patients , 23 sera from grass pollen allergic patients and 25 baker &# 39 ; s asthma patients ( constantin et al , unpublished , data not shown ). the analysis of igg subclass reactivities to the clone 10 - derived allergen in the group of baker &# 39 ; s asthma patients showed the presence allergen - specific igg 1 and to a lower extent of allergen - specific igg 4 levels both indicative of a th 2 response whereas no relevant igg 2 and igg 3 reactivity specific for the clone 10 - derived allergen was detected ( fig4 ). to study the allergic activity of ige antibodies specific for the serine proteinase inhibitor - like allergen rbl cells expressing the human fcεri were loaded with serum ige from patients with and without specific ige antibodies and subsequently exposed to the allergen ( fig5 ). rbl cells loaded with serum ige from patient # 1 showed the strongest degranulation upon allergen exposure ( 51 % of total β - hexosaminidase release ). a lower degranulation was obtained with rbl cells that had been loaded with ige from patients # 4 and # 12 ( 22 % and 19 %, respectively ) which corresponded with the intensity of ige recognition in the dotblots ( fig3 ). almost no degranulation was observed when rbl cells were loaded with serum from a non - allergic person ( fig5 : nc ). the major timothy grass pollen allergen rphl p 1 induced strong degranulation in rbl cells loaded with serum ige from patient # 12 and mild degranulation when rbl cells were loaded with sera from patients # 1 and # 4 ( fig5 ). in fact , patients # 1 , # 4 and # 12 suffered also from grass pollen allergy ( table i ). the serine proteinase inhibitor - like allergen accumulates in wheat seeds during maturation rabbit antibodies , specific for clone 10 - derived allergen , were used to investigate the expression of the protein during wheat seed maturation ( fig6 ). nitrocellulose sheets containing extracts from wheat seeds collected at different time points of seed maturation were probed with specific rabbit antibodies and the pre - immune ig . the clone 10 - specific antibodies reacted with a protein of 40 kda , representing a tetramer of the serine proteinase inhibitor - like allergens ( fig6 ) ( 11 ). the expression of the protein became detectable in 15 days old seeds and continued to increase during further maturation of seeds ( fig6 ). no immunoreactivity was found when the blots were incubated with the pre - immune ig from the same rabbit ( fig6 ). the serine proteinase inhibitor - like allergen is preferentially detected in wheat seeds . when we compared pollen and seeds , we found that the serine proteinase inhibitor - like allergen is preferentially expressed in seeds ( fig7 ) whereas only a weak signal was obtained at approximately 65 kda in wheat pollen extract . the panallergen profilin was detected with rabbit anti - wheat seed profilin antibodies in wheat seeds and pollen ( fig7 : # 123 ). no reactivity was found with pre - immune ig ( fig7 ). next we used the serine proteinase inhibitor - like allergen - specific antibodies to search for cross - reactive structures in rice , maize , common bean and potato for which homologous proteins have been described with a sequence identity of 50 % ( bean ), 49 % ( maize , rice ) and 33 % ( potato ) ( table iii ). the serine proteinase inhibitor - like allergen was detected again as tetramer in wheat seeds , a band of approximately 23 kda was detected in rice but no reactivity was found in maize , common bean or potato ( fig8 c ) although comparable amounts of each extract had been subjected to sds - page ( fig8 a ). no reactivity was observed when the blots were incubated with the pre - immune ig ( fig8 b ). localization of the serine proteinase inhibitor - like allergen in the aleurone layer and between starch granules of a wheat grain by immunogold electron microscopy fig9 a shows an ultra - thin section through a wheat grain at low magnification in the transmission electron microscope . three major morphological components of the grain are visible : an outward multi - layered fruit and seed coat ( c ), the aleuron layer ( al ) and the beginning of the voluminous interior of the grain , the starchy endosperm ( se ). the rectangle marks an area comparable to the area shown in b . fig9 b displays the border between an aleuron cell and the adjoining starchy endosperm at higher magnification . the aleuron cell is filled with aleuron grains ( ag ) ( protein vacuoles ) which are surrounded by small lipid vesicles ( l ). both components are embedded in the cytoplasmic matrix of the cell . the starchy endosperm consists of starch granules with variable size , closely packed just leaving small interspaces of amorphous cytoplasmatic material . the rectangles indicate areas shown in high magnification in c , d and e , f , respectively . fig9 c shows the localization of clone 10 - derived allergen in an aleuron cell using abs raised against wheat protein 10 . gold particles ( arrows ) indicate the presence of wheat protein 10 predominantly in the cytoplasmatic matrix between cell organelles but also in the peripheral parts of the lipid vesicles ( l ). in the starch region , wheat protein 10 is associated with the amorphous cytoplasmatic material ( cy ) in between the starch granules ( sg ) ( fig9 e ). control experiments with preimmune abs showed a very low degree of non - specific labelling ( fig9 , d and f ). example 2 shows the identification and characterization of six ige reactive wheat seed allergens named clones # 10 , # 37 , # 38 , # 112 , # 123 and # 126 . furthermore example 2 shows a method for expression and purification of recombinant allergens of said clones . biological materials , patients &# 39 ; sera wheat seeds from triticum aestivum cv . michael were obtained from the österreichische agentur für gesundheit and ernährungssicherheit gmbh and planted in a glasshouse . immature seeds were harvested directly into liquid nitrogen after a period of 25 days until pollination started and stored at − 80 ° c . until use . sera were obtained from 24 patients suffering from bakers &# 39 ; asthma ( table iv ). baker &# 39 ; s asthma was diagnosed on the basis of case history , total serum ige levels , ige specific for wheat and rye by cap - feia system ( phadia , uppsala , sweden ) and after specific inhalation challenge tests ( 1 ) ( fig1 ). serum from grass pollen allergic patients had been analyzed for total serum ige levels and ige specific timothy grass pollen by cap - feia system ( phadia ) and food allergic patients to wheat have been characterized as described previously ( 2 ) ( fig1 ). clinical data of these patients are summarized in table v . for control purposes serum from a non - allergic individual was included in the experiments . total rna was extracted from wheat seeds stored at − 80 ° c . according to yeh [ 3 ]. then the rna pellet was dissolved in guanidinium isothiocyanate buffer ( 4m guanidinium isothiocyanate , 0 . 83 % v / v 3m sodium acetate , ph 6 , 0 . 11m β - mercaptoethanol ) and purified by a cesium chloride density gradient ultracentrifugation . poly - a + rna was isolated by oligo - dt cellulose affinity chromatography ( nucleo trap mrna ; machery - nagel ) and double stranded cdna was synthesized via a cdna synthesis kit ( cdna synthesis system ; roche diagnostics , mannheim , germany ). after methylation with ecori methylase ( new england biolabs , beverly , mass . ), ecori linkers ( new england biolabs ) were added to the cdna . linkered cdna was digested with ecori ( roche diagnostics ). digested linkers were removed with a nick column ( pharmacia biotech , uppsala , sweden ) and the cdna was ligated into λgt11 arms ( stratagene , la jolla , calif ., usa ). the ligation product was packed in vitro ( gigapack iii gold cloning kit , stratagene ) resulting in a λgt11 expression cdna library with 2 . 43 × 10 6 pfu ( plaque forming units ). isolation and characterization of ige - reactive clones from a wheat seed cdna library e . coli y1090 were infected with 7 × 10 5 pfu of recombinant phages and immunoscreened with serum ige of four patients (# 1 , # 2 , # 5 , # 13 ) suffering from bakers &# 39 ; asthma as described [ 5 ]. six ige - reactive phage clones were selected for further re - cloning and their dna was pcr - amplified using platinum pcr supermix ( invitrogen , life technologies ) with λgt11 primers and sequenced ( mwg , ebersberg , germany ). the obtained sequences were compared with sequences submitted to the genbank database at the national center for biotechnology information ( ncbi ). the coding region of the clones were amplified by pcr using primers listed in table vi . the pcr products were subcloned into an acceptor vector ( novagen , madison , wis .) and sequenced again ( mwg ). then the insert containing acceptor vectors were digested with ndei and ecori ( roche diagnostics ), the inserts were gel - purified ( promega , madison , wis ., usa ) and subcloned into the expression plasmid pet 17b ( novagen ). the dna sequences were confirmed by sequencing both dna strands ( microsynth , balgach , ch ). the pet 17b - insert constructs were transformed into e . coli bl21 ( de3 ) ( stratagene ) and grown in luria broth ( lb ) medium containing 100 mg / l ampicillin at 37 ° c . to an od ( 600 nm ) of 0 . 8 - 1 . protein expression was induced by addition of isopropyl β - d - thiogalactopyranoside ( iptg ) to a final concentration of 0 . 5 mm and growing the bacteria for additional 3 h . then bacteria were harvested by centrifugation and homogenized in 25 mm imidazole , ph 7 . 5 , 0 . 1 % ( v / v ) triton x - 100 with an ultraturrax ( ika , stauffen , germany ). dna was digested by addition of dnase i , stirred for additional 10 min at 20 ° c ., stopped with 200 μl of 5m nacl and then centrifuged at 4 ° c . ( 6000g , 20 min ). clone 10 - derived allergen was purified from the inclusion body - containing pellet under denaturing conditions over ni - nta resin affinity columns according to the qiaexpressionist handbook , protocol 17 ( qiagen , hilden , germany ). fractions containing the recombinant allergen were pooled and dialyzed against 10 mm nah2po4 ph 7 . 5 . recombinant proteins # 37 , # 38 , # 112 , # 123 and # 126 were purified from the pellet of the bacterial cell lysate under native conditions over ni - nta resin affinity columns according to the qiaexpressionist handbook , protocol 12 ( qiagen ). fractions containing the recombinant proteins were pooled and dialyzed against 10 mm nah2po4 ph 7 . 5 . protein samples were analyzed for purity on a 14 % sds - page gel and visualized by coomassie brilliant blue staining . the protein concentrations were determined with micro bca protein assay kit ( pierce , rockford , ill .). example 3 shows an allergen micro - array revealing that recombinant wheat seed allergens , prepared as described in example 2 , are specifically recognized by serum ige from baker &# 39 ; s asthma patients but not from patients with food allergy to wheat or grass pollen allergy . hence the recombinant allergens are specific for respiratory allergy to wheat flour . moreover example 3 shows the use of timothy grass pollen marker allergens phl p 1 and phl p 5 and wheat pollen in the array for improvement of in vitro diagnosis of respiratory allergy . sera were obtained from spanish patients suffering from baker &# 39 ; s asthma ( b1 - b23 ), austrian ( f1 - f26 ) and german patients ( f27 - f38 ) suffering from wheat induced food allergy and austrian patients ( g1 - 017 ) suffering from grass pollen allergy . patients were selected according to a positive case history , specific inhalation challenge tests for confirmation of a clinically relevant sensitization in case of baker &# 39 ; s asthma ( 1 ) and double blind placebo controlled food challenge ( dbpcfc ) in infant patients with food allergy to wheat . serum from all patients has been analyzed for total serum ige levels and ige specific for wheat flour and timothy grass pollen by the cap - feia system ( phadia , uppsala , sweden ). demographic , clinical and serological data of these patients are summarized in table vii , viii and ix . for control purposes , sera from healthy individuals were included in all experiments . recombinant wheat proteins # 10 , # 37 , # 38 , # 112 , # 123 and # 126 derived from a cdna library by screening with sera from baker &# 39 ; s asthma patients were expressed in e . coli and purified as described ( 12 ). wheat pollen were purchased from allergon ( vällinge , sweden ) and recombinant phl p 1 , phl p 5 , phl p 7 , phl p 12 from biomay ( vienna , austria ). triticum aestivum pollen ( 500 mg ) was extracted at 4 ° c . over night in 5 ml pbs , 2 mm edta , 1 mm pmsf . after centrifugation for 1 h at 13 , 000 × g 4 ° c ., the protein concentration of the supernatant was determined with micro bca protein assay kit ( pierce , rockford , ill .) and aliquots were stored at − 20 ° c . until use . wheat pollen extract , 1 - 1 . 5 ng / spot , recombinant and purified wheat proteins and recombinant grass pollen allergens , 0 . 1 - 0 . 15 ng / spot , were spotted with a nano plotter np2 ( gesellschaft für silizium - mikrosysteme mbh , groberkmannsdorf , germany ) on nitrocellulose membranes that were attached to microscope glass slides as described ( 13 ). purified human ige was used as a position marker ( 14 ). the spotted microarrays were pre - washed with 30 μl assaybuffer ( weak phosphate buffer , ph 7 . 5 ) and incubated with 30 μl undiluted sera from allergic patients or controls . after washing with 30 μl assaybuffer bound ige antibodies were detected with 20 μl fluorophore - conjugated anti - ige antibody and fluorescence intensities ( fi ) were measured at a wavelength of 635 nm ( genepix 4000b fran axon ). the cut off level was set to fi = 300 based on values obtained with human serum albumin . we analyzed patients suffering from baker &# 39 ; s asthma , wheat - induced food allergy or grass pollen allergy . the group of baker &# 39 ; s asthma patients consisted of 23 persons ( 4 females , 19 males : mean age 39 years , range 22 - 60 years ) with occupational exposure to wheat flour . ninety - one percent of the baker &# 39 ; s asthma patients suffered from asthma due to inhalation of wheat flour and 94 % complained about symptoms of rhinoconjunctivitis . a sensitization to other respiratory allergen sources ( e . g ., cat , cockroach , dog , grass pollen , horse , house dust mites , moulds and / or olive pollen ) was found in 70 % of the baker &# 39 ; s asthma patients and 48 % of the patients suffered from grass pollen allergy ( table vii ). interestingly , none of the baker &# 39 ; s asthma patients exhibited allergy to food and symptoms were confined to respiratory manifestations . the group of patients suffering from wheat - induced food allergy comprised 38 individuals ( 25 females , 13 males : mean age 13 years , range 0 . 5 - 65 years ) ( table viii ). again we found that 71 % are sensitized to other respiratory allergen sources ( e . g ., birch pollen , grass pollen , house dust mites and mugwort pollen ) and 58 % were also sensitized to food allergens ( e . g ., carrot , cow &# 39 ; s milk , hazelnut , hen &# 39 ; s egg , malt , nuts , orange , plum , rice , soybean , celery , seafood , shrimps and / or spices ) ( table viii ). ige - mediated sensitization to grass pollen was found in 55 % of these patients . the symptoms of these patients varied from respiratory symptoms ( e . g ., asthma , bronchitis , cough , conjunctivitis , dyspnea , nasal congestion , rhinorrhoe , rhinoconjunctivitis ) to gastrointestinal symptoms ( e . g ., abdominal pain , diarrhoea , flatulence , sore throat and vomiting ) and cutaneous symptoms ( e . g ., eczema , pruritus and urticaria ) ( table viii ). the group of grass pollen allergic patients consisted of 17 patients ( 5 females , 12 males : mean age 13 years , range 0 . 5 - 65 years ). seventy one percent of these patients suffered also from allergy to other respiratory allergen sources ( e . g ., birch pollen , cat , dog , house dust mites , rabbit and mugwort pollen ) ( table ix ). according to serology 65 % exhibited ige reactivity to wheat flour and 23 % contained ige against other food allergens . however , only one patient suffered from urticaria and no symptoms of food allergy could be recorded for these patients . respiratory symptoms such as asthma , conjunctivitis , dyspnea , rhinoconjunctivitis and rhinitis dominated in the grass pollen allergic patients . six recombinant wheat seed allergens designated as # 10 , # 37 , # 38 , # 112 , # 123 and # 126 were spotted onto nitrocellulose - coated glass slides ( fig1 , a ). the recombinant wheat proteins were expressed in escherichia coli based on cdnas which had been isolated from a wheat seed cdna library with sera from baker &# 39 ; s asthma patients as described ( 15 ). according to homology with sequences deposited in the ncbi database the wheat allergens can be described as follows : # 10 has a 96 % homology to a triticum aestivum subtilisin - chymotrypsin inhibitor wsci precursor ( accession no . gi | 122065237 ), # 37 is a t . aestivum thioredoxin h ( accession no . gi | 27461140 ), # 38 is a t . aestivum glutathione transferase ( accession no . gi | 20067419 ), # 112 has a 99 % homology with a t . aestivum 1 - cys - peroxyredoxine ( accession no . gi | 34539782 ), # 123 has a 96 % homology with t . aestivum profilin ( accession no . gi | 1346803 ) and # 126 has a 69 % homology with hordeum vulgare dehydrin 11 ( accession no . gi | 4105101 ). in addition to the recombinant wheat allergens , wheat pollen extract , recombinant timothy grass pollen allergens ( rphl p 1 , rphl p 5 , rphl p 7 and rphl p 12 ) ( 16 - 19 ) and ige were also spotted onto the slides ( fig1 , a ). representative images of allergen microarrays after incubation with sera are shown in fig1 , b - c . on the chip probed with serum from a non - allergic person showed no reactivity with any allergen could be detected and only the spotted ige markers were detected with the anti - ige conjugate ( fig1 , b ). figures in 12 , c show representative images of microarray chips incubated with sera from a baker &# 39 ; s asthma patient ( 1 ), a patient suffering from wheat - induced food allergy ( 2 ) and a grass pollen allergic patient ( 3 ). the baker &# 39 ; s asthma patient ( 1 : table i : b4 ) shows strong ige reactivity to recombinant wheat allergens # 10 and weak reactivities to # 126 and # 112 . for the food allergic patient ( 2 : table viii : f26 ) strong ige binding to recombinant profilin from wheat # 123 and timothy grass pollen rphl p 12 and weak binding to wheat pollen extract and rphl p 1 was observed . the image of the chip from a grass pollen allergic patient ( 3 : g16 ) shows strong signals to rphl p 5 and rphl p 12 and weaker signals with wheat profilin # 123 , wheat pollen extract and rphl p 1 . identification of wheat seed allergens specifically recognized by ige antibodies from baker &# 39 ; s asthma patients ninety one percent of the baker &# 39 ; s asthma patients were positive in the wheat flour cap ( table vii ). using spotted wheat allergens the ige reactivity profile for 62 % of the baker &# 39 ; s asthma patients could be established . allergens # 126 ( 30 %), # 10 ( 26 %), # 123 ( 22 %) and # 122 ( 17 %) were the most frequently detected components whereas # 37 and # 38 reacted only with 4 % of the sera ( fig1 , a ). interestingly , the two patients who were negative in the wheat flour cap ( b5 , b18 : table vii ) reacted with allergen # 126 . forty eight percent of the baker &# 39 ; s asthma patients showed ige reactivity to timothy grass pollen extract and each of these patients was also diagnosed with a combination of spotted rphl p 1 and rphl p 5 . recombinant phl p 12 ( 35 %) was always stronger and more often recognized than wheat profilin # 123 ( 22 %). the cross - reactive pollen allergen rphl p 7 reacted with 13 % of the sera . recombinant wheat seed allergens recognized by baker &# 39 ; s asthma patients are not targets for ige antibodies of patients suffering from wheat - induced food allergy all patients with food allergy to wheat were positive in the wheat flour cap but the recombinant wheat proteins were hardly recognized ( 5 %) ( fig1 , b ). the two patients who were positive to cross - reactive timothy grass pollen profilin , had also a positive signal to wheat profilin # 123 in the microarray . fifty five percent of patients with wheat induced food allergy showed ige reactivity in the phleum cap but just 18 % were positive to wheat pollen extract on the chip and 26 % in the combination of spotted rphl p 1 and rphl p 5 . spotted rphl p 1 and rphl p 5 alone were recognized by 18 % and 16 % of the sera respectively , the cross - reactive allergens rphl p 7 and rphl p 12 reacted with 13 % and 26 % of the sera . rphl p 1 and rphl p 5 are diagnostic marker allergens for grass pollen allergy whereas profilin is recognized by baker &# 39 ; s asthma and wheat food allergic patients sixty - five percent of grass pollen allergic patients were positive in the wheat flour cap . out of the recombinant wheat proteins tested in the microarray , only recombinant wheat protein # 123 was recognized by 23 . 5 % of the patients ( fig1 , c ). each of these patients were also positive to cross - reactive timothy grass pollen profilin rphl p 12 . but rphl p 12 was always stronger and more often ( 35 %) recognized than # 123 . all patients suffering from grass pollen allergy were positive on the timothy grass pollen cap and in the combination of spotted rphl p 1 and rphl p 5 . recombinant phl p 1 alone was more often recognized ( 94 %) than rphl p 5 ( 88 %) by patients with grass pollen allergy . wheat pollen extract reacted with 82 % of the sera and cross - reactive rphl p 7 with 29 %. all grass pollen allergic patients and baker &# 39 ; s asthma patients with grass pollen allergy were positive in the combination of rphl p 1 and rphl p 5 in the microarray and in the phleum cap ( fig1 , a ; c ). these findings are in agreement with previously published data showing that grass pollen allergy can be diagnosed by ige reactivity to rphl p 1 and rphl p 5 ( 20 , 21 ). however , among patients with wheat induced food allergy only 26 % reacted with rphl p 1 and rphl p 5 in the microarray , but more than twice as many ( 55 %) were positive in the phleum cap ( fig1 , b ). furthermore , those patients reacting with grass pollen extract in the cap system and with timothy grass pollen profilin ( rphl p 12 ) in the microarray were also positive to wheat profilin ( 22 ) due to cross - reactivity of plant profilins ( 23 ). nevertheless , frequency and intensity of ige recognition was always stronger to rphl p 12 than to wheat profilin ( 22 ). the analyte is immobilized to a solid support , such as immunocap ( phadia , uppsala , sweden ). serum samples from at least three representative human patients sensitized to the allergen and showing ige reactivity to that allergen are incubated for 3 h at room temperature with the allergen at a final concentration of 100 μg / ml and , in parallel as negative controls , with buffer alone and the non - allergenic maltose binding protein ( mbp ) of e . coli . the samples are then analysed for ige binding to immunocap ( phadia , uppsala , sweden ) tests carrying immobilized analyte to study whether preincubation with allergen specifically inhibits or significantly lowers ige binding . demographic , clinical and serological characteristics of patients suffering from bakers &# 39 ; 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s asthma . unpublished . 16 . laffer s , valenta r , vrtala s , susani m , van ree r , kraft d , et al . 1994 . complementary dna cloning of the major allergen phl p i from timothy grass ( phleum pratense ); recombinant phl p i inhibits ige binding to group i allergens from eight different grass species . j allergy clin immunol 94 : 689 - 98 . 17 . vrtala s , sperr w r , reimitzer i , van ree r , laffer s , muller w d , et al . 1993 . cdna cloning of a major allergen from timothy grass ( phleum pratense ) pollen ; characterization of the recombinant phl pv allergen . j immunol 151 : 4773 - 81 . 18 . niederberger v , hayek b , vrtala s , laffer s , twardosz a , vangelista l , et al . 1999 , calcium - dependent immunoglobulin e recognition of the apo - and calcium - bound form of a cross - reactive two ef - hand timothy grass pollen allergen , phl p 7 . faseb j 13 : 843 - 56 . 19 . valenta r , ball t , vrtala s , duchene m , kraft d , scheiner 0 . 1994 . cdna cloning and expression of timothy grass ( phleum pratense ) pollen profilin in escherichia coli : comparison with birch pollen profilin . biochem biophys res commun 199 : 106 - 18 . 20 . kazemi - shirazi l , niederberger v , linhart b , lidholm j , kraft d , valenta r . 2002 . recombinant marker allergens : diagnostic gatekeepers for the treatment of allergy . int arch allergy immunol 127 : 259 - 68 . 21 . andersson k , lidholm j . 2003 . characteristics and immunobiology of grass pollen allergens . int arch allergy immunol 130 : 87 - 107 . 22 . sampson h a . 1999 . food allergy . part 2 : diagnosis and management . j allergy clin immunol 103 : 981 - 9 . 23 . radauer c , willerroider m , fuchs h , hoffmann - sommergruber k , thalhamer j , ferreira f , et al . 2006 . cross - reactive and species - specific immunoglobulin e epitopes of plant profilins : an experimental and structure - based analysis . clin exp allergy 36 : 920 - 9 .