Mapping of IgE-binding regions on recombinant Cyn d 1, a major allergen from Bermuda Grass Pollen (BGP)
© Tiwari et al; licensee BioMed Central Ltd. 2009
Received: 22 September 2008
Accepted: 02 February 2009
Published: 02 February 2009
Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) is an important source of seasonal aeroallergens in warm tropical and sub-tropical areas worldwide. Improved approaches to diagnosis and therapy of allergic diseases require a thorough understanding of the structure and epitopes on the allergen molecule that are crucial for the antigen-antibody interaction. This study describes the localization of the human IgE-binding regions of the major group 1 pollen allergen Cyn d 1 from Bermuda grass.
A cDNA library was constructed from Bermuda grass pollen (BGP) using a Lambda gt11 expression vector. The gene encoding the Cyn d 1 allergen was isolated by screening the library with a mouse monoclonal antibody raised against grass group 1 allergen. In order to characterize the IgE epitopes on Cyn d 1, seven overlapping fragments and three deletion mutants were cloned and over-expressed in E. coli. The recombinant fragments and deletion mutants were evaluated for their comparative IgE reactivity with sera of non atopic individuals and grass pollen allergic patients by ELISA and a dot-blot assay.
Analysis of IgE binding regions by overlapping fragments and deletion mutants identified two major allergenic regions corresponding to amino acids 120–170 and 224–244. Deletion of either or both regions led to a significant reduction in IgE binding, emphasizing the importance of the C-terminal region on Cyn d 1 in epitope-IgE interaction.
Anti-Cyn d 1 IgE antibodies from allergic human sera recognize two epitopes located at the C-terminal end of the molecule. These data will enable the design of improved diagnostic and therapeutic approaches for BGP hypersensitivity.
It is estimated that up to 20% of the population in developed countries suffers from atopic diseases caused by airborne allergens derived from grass and tree pollen, house dust mites and animal dander, which has a significant impact on quality of life and economic consequences [1, 2]. The role of grass pollen allergens in triggering immunoglobulin E (IgE)-mediated type I allergic diseases, such as allergic rhinitis, conjunctivitis, and bronchial asthma is very well established [3, 4]. Epidemiologic studies have revealed that allergies to Bermuda grass (Cynodon dactylon; subfamily Chloridoideae) mainly affect people in warm tropical and sub-tropical areas of the world, such as southwestern United States, South Africa and northern and central Australia. Furthermore, approximately 27% of asthmatic patients in Taiwan are reported to have a hypersensitive response to crude Bermuda grass pollen (BGP) extract .
Cyn d 1, the major allergen of BGP, is the most abundant protein component of BGP, comprising 15–20% of crude pollen extract. It dominates the human IgE response with 87% of individuals allergic to BGP showing positive reaction to this 32 kDa protein [6, 7]. Clinical evidence of cross-reactivity among grass pollens has suggested that diagnosis and effective immunotherapy can be achieved with a limited number of grasses, although the selection of species to treat against is based on regional prevalence and taxonomic relationship. Grass group 1 allergens, with molecular weights in the range of 31–35 kDa, are recognized as one of the most prominent allergenic components of pollen extracts. In recent years, the genes encoding Group 1 allergens for several grasses have been cloned and expressed using molecular biology techniques [8, 9]. When translated, the cDNA sequences predict proteins of approximately 240 amino acid residues and molecular weights of about 26 kDa. They share an N-glycosylation site at amino acid position 9 and carry glycans that account for 4–5% of their molecular weight. These glycoproteins are located in the cytoplasm of the pollen grain and are rapidly released when hydrated upon contact with moist mucosal surfaces. A characteristic feature of group 1 allergens is the presence of seven strictly conserved cysteine residues, located mainly in the N-terminus of the protein [10–12].
The first full length cDNA coding for Cyn d 1 was identified by Smith et al  followed by Chang et al . Au and colleagues have suggested the existence of two groups of Cyn d 1, the acidic and the basic. The Cyn d 1 cDNAs belonging to the basic group have N-terminal sequences of AI GDKPNITATYGSK WLE, while the sequences of acidic isoallergens showed substitutions of M, D, L, D for I, S, K, E (italicised in the aforementioned sequence), respectively .
Comparison of amino acid sequences of group 1 allergens reveals a high degree of sequence similarity, which highlights a possible basis of allergenic cross-reactivity observed among group 1 allergens when investigated by inhibition techniques among taxonomically related grasses [14–16]. However, earlier studies on allergens from BGP have shown that BGP allergens share minimal IgE cross-reactivity with pollen from Poaceae sub-family grasses, such as Dactylis glomerata, Lolium perenne, Festuca pratensis, Phleum pratense and Poa pratensis when tested with crude pollen extracts. These findings suggest the presence of unique IgE epitopes in BGP allergens [17, 18]. In a recent study, twenty specific anti-Phl p 1 monoclonal antibodies (MAbs) were produced from BALB/c mice immunized with natural Phl p 1. When tested for specificity with thirteen different grass pollen extracts, eighteen to nineteen anti-Phl p 1 MAbs recognized the homologous allergen in pollen extracts from grasses of the Poeae tribe while only four MAbs recognized the group 1 allergen from Cynodon dactylon . These studies further suggest that the antigenic regions of Cyn d 1 in BGP may be different from Poeae grasses, and as a result, individuals allergic to BGP may require separate diagnosis and therapy.
Recombinant allergens are useful tools for structural analysis, including that of IgE binding epitopes, and may provide a better understanding of the allergic immune response. Although allergenic properties and IgE epitopes from Poaeae grasses have been studied extensively [20–22], information on the IgE epitopes of Cyn d 1 is still lacking. Studies of this nature are important because a sizable population is exposed and sensitized to BGP, and it seems to be immunologically distinct from other clinically important allergenic grasses. In light of the prevalence of BGP in warm tropical regions and its role as a respiratory allergen, we focussed on identification of the immunodominant regions of the major BGP allergen Cyn d 1. To locate the relevant IgE binding regions on Cyn d 1, a series of seven overlapping recombinant peptides and three deletion mutants generated from the cDNA of Cyn d 1 were investigated for their IgE binding capacity. We report the presence of at least two major IgE binding regions located within the C-terminus of Cyn d 1.
Pollen extract and patient sera
Human allergic sera were obtained from patients with a clinical history and positive skin test reaction to Bermuda grass pollen. Normal control serum samples were obtained from healthy non-atopic subjects with a negative skin test to BGP. BGP was obtained as dry, non-defatted pollen from Greer Laboratories (North Carolina, USA). Pollen extracellular proteins were extracted by incubation in PBS (10 mM Na2HPO4, 1.8 mM KH2PO4, 140 mM NaCl, 2.7 mM KCl, pH 7.4) for 10 min at room temperature with periodic stirring. The extract was filtered through a Whatman No. 1 filter and centrifuged at 5000 g for 30 min at 4°C to separate the supernatant containing the secreted protein. The serine protease inhibitor phenylmethylsulphonyl fluoride (PMSF [0.05% v/v]) was added to the extract to prevent protein degradation due to protease activity. Both sera and pollen were stored at -20°C until use.
Isolation of messenger RNA from Cynodon pollen
Cynodon dactylon pollen was purchased from Greer Laboratory (North Carolina, USA). Total RNA was isolated from 500 mg pollen using a slightly modified guanidine hydrochloride method described by Logemann and co-workers . Pollen grains were finely ground in liquid nitrogen, and mixed in 5 mL of extraction buffer (7.5 M guanidine HCl in 0.1 M β-mercaptoethanol, 0.5% w/v lauryl sarcosinate and 25 mM sodium citrate) prepared in diethyl pyrocarbonate (DEPC) treated water. The extract was centrifuged at 12,000 g for 20 min. The supernatant was collected and purified twice with equal volume of phenol: chloroform: isoamylalcohol (25:24:1 v/v) followed by centrifugation. The RNA was treated with DNase and the quality was analysed by agarose gel electrophoresis. Aliquots were stored at -70°C. Poly (A) messenger RNA was affinity purified using oligo-dT cellulose (Microfast trak Invitrogen), following the manufacturer's instructions.
Construction and immunoscreening of the Bermuda grass pollen cDNA library
Double-stranded cDNA was synthesized from purified mRNA using a synthesis kit (Pharmacia LKB, Uppsala, Sweden) according to manufacturer's instructions. The cDNA was ligated to an Eco R1/Not 1 adaptor, cloned into a Lambda gt 11 vector (Amersham), and packaged into bacteriophages (Gigapak Gold III Stratagene) to construct a cDNA expression library. E. coli Y1090 competent cells were transduced with in vitro packaged phages, which resulted in a primary library with 98% recombinant clones. The cDNA library was screened with anti-Cyn d 1 mouse mAb 3A2, which binds Cyn d 1. Screening of the cDNA library was carried out by taking plaque lifts and probing with mAb 3A2. Briefly, lambda-gt11 phage with the cDNA inserts were used to infect E. coli Y1090 cells, plated onto LB plates (density 200–300 plaques/plate) containing 100 μg/mL ampicillin and incubated at 42°C for 4 h. The plates were overlaid with nitrocellulose filters (Hybond-C extra, Amersham, U.K.) soaked with 1 mM IPTG (isopropyl-β-D-thiogalacto-pyranoside) and incubated at 37°C in order to induce the expression of fusion protein. Two lifts were taken from each plate at 4-hour intervals. Subsequently, the membranes were washed with PBS-Tween20, blocked with 10% skim milk powder in PBS and incubated with mAb 3A2 (1:1000 in 1% BSA/PBS) for 5–6 hours. The membranes were washed with PBS-0.05% Tween20 and incubated with alkaline phosphatase conjugated anti-mouse IgG, 1:1000 (Sigma) at room temperature for 2 h. The positive clones were selected by colorimetric development of membranes with 0.4 mmol/L nitroblue tetrazolium (Boehringer, Mannheim, Germany), 0.4 mmol/L 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Astral Scientific, NSW, Australia). The plaques for positive clones were excised and further verified by secondary screening.
Isolation of Lambda DNA of positive clones
Lambda lysates were prepared by incubating an isolated plaque with 500 μL of plating E. coli Y1090 cells for 15 min, after which the volume was made up to 5 mL with LB containing 100 μg/mL ampicillin, 5 mM calcium chloride, 0.4% w/v maltose and the culture incubated at 37°C, with shaking, for 5 h or until the bacterial cells were lysed; thereafter 500 μL of chloroform was added to each culture and incubated for a further 15 min. Bacterial debris was removed by centrifugation at 8000 g for 10 min. The lambda lysate was treated with DNase (10 μg/ml) and RNase (20 μg/ml) at 37°C for 30 minutes to remove traces of bacterial genomic DNA and RNA. Lambda DNA was isolated twice from liquid lysates with an equal volume of phenol: chloroform: isoamylalcohol (25:24:1 v/v) and the suspension gently mixed for 20 minutes followed by centrifugation at 12,000 g for 20 minutes at 4°C. Final extraction was carried out with equal volume of chloroform: isoamylalcohol (24: 1). The supernatant was transferred to a fresh tube and RNA was precipitated with 1/10 volume of 3 M sodium acetate and equal volume of cold isopropanol. The mixture was stored at -20°C and DNA pellet was recovered by centrifugation at 4°C for 20 minutes at 12,000 g. The pellet was washed with 70% ethanol, vacuum dried and dissolved in TE buffer (pH 7.4).
Cloning and Sequencing of positive cDNA clones
The cDNA inserts of various clones were identified by PCR with lambda-gt11 forward and reverse primers on purified DNA template. The resulting PCR products were gel purified (Jetsorb Kit, Astral Scientific) and sub-cloned into pGEM-T vector (Promega). Identity of the clones was verified by automated sequencing (Applied Biosystems, USA) using T7 and SP6 DNA sequencing primers. The nucleotide sequence and deduced amino-acid sequences were compared by FASTA with those in the GeneBank database. Sequence alignments were performed with the CLUSTAL W package.
PCR cloning of rCyn d 1 and its derived, overlapping fragments and deletion mutants
Sequence of primers used for cloning rCyn d 1 and of Cyn d 1 fragments and deletion mutants in expression vector pQE30Xa.
Sequence (5' – 3')
Expression and purification of recombinant Cyn d 1 and of Cyn d 1 fragments and deletion mutants
Briefly, a 250 mL volume of LB broth containing 50 mg/ml kanamycin and 100 mg/mL ampicillin (Sigma Aldrich) was seeded with 5 ml overnight culture of E. coli M15 cells (Qiagen) harbouring the expression plasmid with either the entire Cyn d 1 cDNA, or the seven overlapping fragments and three deletion mutants. The cultures were grown at 37°C to log phase with an optical density of A600 = 0.6. Isopropyl-βD-thiogalactoside (IPTG) (Progen) was added to a final concentration of 1 mM to induce expression of the fusion protein. Following incubation at 37°C for a further 3 hours, bacterial cells were harvested by centrifugation at 10,000 g for 20 minutes. The cell pellet from 1 mL of culture was resuspended in 100 uL of reducing SDS sample buffer and subjected to SDS-PAGE to evaluate expression levels. Cells were harvested from the culture samples by centrifugation and stored at -20°C. The cells were lysed by repeated freeze thaw cycles in liquid nitrogen. The partially lysed cells were then resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH 8.0). Lysozyme (Sigma, Aldrich) at 1 mg/ml was added to the resuspension, which was incubated on ice for 30 minutes for further cell lysis. RNase A (Sigma, Aldrich) at 10 μg/ml and DNase (Sigma, Aldrich) at 5 μg/ml was added to the lysate and incubated for 15 minutes to remove bacterial RNA and DNA and to reduce the viscosity of the cell lysate. The lysate was then centrifuged at 10,000 g for 20 min at 4°C. The supernatant was collected and added to 1 mL TALON Ni-NTA resin (Clontech, USA) to allow the resin to bind to the His tagged recombinant protein. The mixture was rocked gently on ice for 1 hour. The resin and supernatant mixture was pipetted into a 1 mL column, (Qiagen) and the column was allowed to settle by gravity before washing with a further two column volumes of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Fusion proteins were eluted from the column with several aliquots of 0.5 mL of elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The purity and yield of the fusion proteins were established on 12.5% Coomassie stained SDS-PAGE gels. Concentrations were measured at OD280.
Human IgE Dot blot analysis
To test the specific IgE-binding capacity of the recombinant peptides and deletion mutants, an immuno dot-blot assay was performed. Varying concentrations (300, 400 and 500 nM) of purified recombinant proteins were applied to a polyvinyl difluoride (PVDF) membrane with a Minifold I Apparatus through vacuum suction. The dot-blots were blocked with PBS (pH 7.4) containing 10% skim milk for 1 h to block free binding sites. Both pooled and individual allergic sera were diluted to 1:5 in PBS and incubated with the dot-blots overnight at 4°C. Incubation of the membranes with the secondary antibody was performed with a 1:1000 dilution of alkaline phosphatase conjugated mouse anti-human IgE (Sigma) for 1 h. The bound human IgE was detected by incubating with 0.4 mmol/L nitroblue tetrazolium (Boehringer, Mannheim, Germany), 0.4 mmol/L 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (Astral Scientific, NSW, Australia) colorimetric substrates, and allowing the color signal on the membranes to develop. The membranes were extensively washed with PBS containing 0.1% Tween 20 after each step throughout the protocol.
Direct ELISA of deletion mutants
The relative binding of IgE to deletion mutants and full-length rCyn d 1 was assessed in an ELISA assay. Briefly, microtiter 96-well plates (Maxisorp, Nunc, Denmark) were coated with 5 ug/well of rCyn d1 and deletion mutant proteins diluted in 0.1 M sodium bicarbonate coating buffer (pH 9.6) for O/N incubation at 4°C. Optimum concentrations of allergen and antibody were determined by checkerboard titration. Non-specific binding sites were blocked by incubating with 200 μl per well of blocking buffer (PBS, 0.05% Tween20 and 1% BSA) for 1 h at room temperature. After five washes with PBS containing 0.05% Tween20, pooled and individual sera from BGP allergic or non-allergic individuals (1:5) were added to each well and incubated O/N at 4°C. The alkaline phosphatase conjugated secondary antibody, mouse anti-human IgE (Sigma), diluted 1:1000 in PBS/0.05% Tween 20/, 1% BSA was added to the wells and incubated for 3 h at room temperature. The bound human IgE on the wells was detected by addition of 0.1% pNPP p-nitro-phenyl phosphate (Sigma) colorimetric substrate. The optical density was measured at 405 nm. PBS buffer and sera from non-allergic subjects were included in the assay as negative controls.
cDNA sequence analysis of Cyn d 1
Expression and purification of rCyn d 1 and its derived fragments and deletion mutants
Dot blot analysis of Cyn d 1 fragments and deletion mutants
IgE reactivity of rCyn d 1 deletion mutants by ELISA
Comparison of patients' IgE binding to rCyn d 1 and deletion mutants.
rCyn d 1
Pool n = 10
Mean ± SEM
54.2 ± 3.5
61.4 ± 2.1
30.6 ± 0.8
In the present study, the cDNA of one of the most relevant environmental aeroallergens, the major Bermuda grass pollen allergen, Cyn d 1 (acidic isoform) was successfully cloned and expressed to produce immunologically active recombinant protein. No previous information was available on the clinically relevant epitopes on Cyn d 1. In the present study, we identified the immunodominant regions on the Cyn d 1 molecule. A collection of overlapping fragments and deletion mutants were expressed along with an N-terminal His tag and purified by Nickel affinity chromatography. The purified proteins were subjected to immuno dot blotting to compare their IgE reactivity with sera from BGP allergic patients. Fragments F1, F2 and F3, covering amino acid residues 1–120, failed to show IgE Ab binding for all sera tested. IgE binding to deletion mutants was weaker compared to intact rCyn d 1. We identified at least two major IgE-binding regions (120–170) and (224–244) by immunodot blot analysis. To further determine the binding of deletion mutants to IgE, a solid-phase ELISA was carried using four individual sera and serum pool from 10 patients with BGP allergy. Both deletion mutants D1 and D2 showed about 40% reduction in IgE binding. Interestingly, deleting 50 residues (120–170) reduced IgE binding to about the same extent as deleting the last 20 residues. The allergenic potency of D3 devoid of both epitopes produced a significant reduction of about 70% in IgE binding in comparison to the whole molecule. These findings clearly indicated that the C-terminal of Cyn d 1 was the major IgE-binding region. Since removal of the two immunodominant regions gave about 70% reduction in IgE reactivity of rCyn d 1, other minor epitopes may be present that account for the remaining 30% reactivity. The data derived from our study are in agreement with IgE epitopes described from other grass group 1 allergens. The involvement of C-terminal region in IgE binding has been reported for the group 1 pollen allergens from several grasses using either synthetic peptide fragments corresponding to linear segments of the allergen or monoclonal antibody based mapping strategies [20–22]. Tamborini and co-workers mapped the IgE binding region of Lol p 1, a major group 1 allergen from Rye grass pollen. They produced three deletion mutants with large deletions in the protein. The first mutant, lacking the N-terminus region (aa 1–88), showed 27% IgE binding, the second mutant, lacking region (aa 132–190), showed 6.3% IgE reactivity, and the third mutant, missing the C-terminus region (aa 116–240), showed 4.5% IgE reactivity as determined by ELISA. A strong reduction in IgE reactivity was measured for the third mutant suggesting the C-terminus as the immunodominant portion of Lol p 1 . A similar approach using gene fragmentation and synthetic peptides was used to map the IgE-binding epitopes of major group 1 allergen of velvet grass pollen Hol l 1  and Phl p 1 from timothy grass . The binding patterns of patients' IgE to small peptides was much weaker than to larger fragments, and 46% of sera showed binding to C-terminus as opposed to only 8% of sera binding to N-terminus region in the case of the Hol l 1 molecule . The findings from B-cell epitope mapping studies of Phl p 1 were also similar to Hol l 1, with the majority of sera recognizing the C-terminal fragment rT147 that comprises amino acids 146 through 240 of Phl p 1 . The IgE-binding regions identified in Cyn d 1 in the current study partially or completely overlap with those identified in other Group 1 allergens where the C-terminal portion has been shown to be immunodominant. The contention that this IgE containing region is representative of group 1 allergens from other grass species, and therefore is relevant for grass pollen allergy in general, is supported by the fact that group 1 allergens show very high sequence similarity (~85%). In addition, this IgE containing regions has been reported to represent prominent IgE epitope bearing region of grass group 1 allergens from other grass species.
The entire population of IgE epitopes for a number of aeroallergens has been shown to be dependent on their structural integrity. One of the approaches used to reduce the anaphylactic potential of any allergen is to disrupt the overall secondary structure of the protein. Since the majority of IgE epitopes are known to be conformational, a collapse in the structure can prevent allergen-specific IgE recognition and cross-linking. In the extensively studied Bet v 1 allergen from birch pollen expressing the molecule as two separate fragments led to complete ablation of IgE reactivity . In case of the fragments generated for Cyn d 1 in this study fragment 4 (aa 1–120), covering the amino-terminal half of the molecule, did not bind IgE while fragment 7 (aa 129–244), covering the C-terminal end, showed positive reaction with IgE. These results suggest that breaking the Cyn d 1 molecule into two halves did not have a significant effect on IgE reactivity. The finding that the majority of the IgE binding was preserved after removing 120 residues from the N-terminal provides further evidence for the C-terminus as a likely candidate representing the major IgE binding region on Cyn d 1.
Based on the findings from this study, the lack of cross reactivity of BGP extract with the Poeae grasses may seem puzzling since most of the IgE antibodies are directed against the C-terminal of the group 1 allergen across the different grasses, including Cyn d 1. One possible explanation for this phenomenon may be because several of the earlier studies were carried out with crude pollen extracts that comprised of a mixture of several allergenic proteins making it difficult to obtain a detailed IgE profile of the different allergenic fractions. Furthermore, three dimensional structure and steric orientation of allergens are crucial for cross-linking of mast cells and basophils-bound IgE antibodies for inducing release of inflammatory mediators. Spatial clustering of IgE-binding sites on the Phl p 1 surface has been shown to be an important factor for its allergenic activity. Results from this study defined a sterically oriented C-terminal fragment of Phl p 1 as the portion with highest allergenic activity . Although group 1 allergens have high sequence identity, point mutations of very few key amino acid residues can drastically alter the protein structure and spatial orientation of the IgE epitope. For example, cysteine residues and disulfide bonds play an important role in IgE binding in the case of grass allergen Lol p 1. Replacement of cysteine residue at position 77 with serine caused an average of 67.2% reduction in human IgE reactivity to this molecule . As discussed previously, even though the major IgE epitope for group 1 allergen is located at the C-terminus, there may be differences in the minor epitopes that could influence the secondary structure of the protein. Alterations of secondary structure may cause constraints on the orientation of the molecule. Small changes in amino acid sequence can alter a protein's structure that, in turn, can affect mast cell induction and basophil activation. Studies have demonstrated that isoforms of the same allergen with very few amino acid differences can exhibit greatly varying potencies to induce an allergic response. For example, a naturally occurring low allergenic isoform has been reported for hazel pollen allergen Cor a 1 . Since the crystal structure of Cyn d 1 protein has not been solved, further studies are needed to finely map the IgE-binding regions of Cyn d 1. Inhibition of IgE binding by synthetic peptides representing homologous amino acids sequences of other Poaeae grasses could provide a better understanding of the precise distribution of IgE epitopes on Cyn d 1 and their role in allergic sensitization. This type of analysis should further elucidate the observed lack of cross reactivity between BGP and pollen from Poae grasses.
In conclusion, we describe here the results of an experimental investigation that led us to identify at least two major IgE binding regions on Cyn d 1, the major allergen from Bermuda grass pollen. We mapped these binding regions to the C-terminal end of the molecule. The location of epitopes within the highly conserved C-terminus of grass group 1 allergens may be a common feature of the IgE response to grass group 1 allergens. Since structural and mutational analyses of allergens can be utilized to develop vaccines for allergy therapy, targeted substitution or deletion of amino acids within the identified epitopes could result in nonanaphylactic allergenic variants that may, in turn, be useful to develop vaccines for BGP allergy sufferers.
This work was supported by Melbourne International Research Scholarship to RT from The University of Melbourne, Australia.
- Jackson M: Allergy: the making of a modern plague. Clin and Exp Allergy. 2001, 31: 1665-1671. 10.1046/j.1365-2222.2001.01264.x.View ArticleGoogle Scholar
- Gerth van Wijk R: Allergy: a global problem. Allergy. 2002, 57: 1097-1110. 10.1034/j.1398-9995.2002.01003.xView ArticleGoogle Scholar
- Andersson K, Lidholm J: Characteristics and immunobiology of grass pollen allergens. Int Arch Allergy Immunol. 2003, 130: 87-107. 10.1159/000069013View ArticlePubMedGoogle Scholar
- Esch R: Grass pollen allergens: in Lockey RF, Bukantz SC (eds). In Allergens and allergen immunotherapy New York, Dekker; 1999, 103-120.Google Scholar
- Kao S, Su SN, Huang SW, Tsai JJ, Chow LP: Sub-proteome analysis of novel IgE-binding proteins from Bermuda grass pollen. Proteomics. 2005, 5: 3805-3813. 10.1002/pmic.200401229View ArticlePubMedGoogle Scholar
- Shen H, Wang SR, Tang RB, Chang ZN, Su SN, Han SH: Identification of allergens and antigens of Bermuda grass (Cynodon dactylon) pollen by immunoblot analysis. Clin Allergy. 1988, 88: 763-774.Google Scholar
- Smith P, Suphioglu C, Griffith IJ, Theriault K, Knox RB, Singh MB: Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen. J Allergy Clin Immunol. 1996, 98: 331-343. 10.1016/S0091-6749(96)70158-6View ArticlePubMedGoogle Scholar
- Dewitt AM, Andersson K, Peltre G, Lidholm J: Cloning, expression and immunological characterization of full-length timothy grass pollen allergen Phl p 4, a berberine bridge enzyme-like protein with homology to celery allergen Api g 5. Clin Exp Allergy. 2006, 36: 77-86. 10.1111/j.1365-2222.2006.02399.xView ArticlePubMedGoogle Scholar
- Suphioglu C, Singh MB: Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen. Clin Exp Allergy. 1995, 9: 853-65. 10.1111/j.1365-2222.1995.tb00028.x.View ArticleGoogle Scholar
- Matthiesen F, Schumacher MJ, Lowenstein H: An immunoelectrophoretic analysis of the allergens of Cynodon dactylon (Bermuda grass) pollen. J Allergy Clin Immunol. 1989, 83: 1124-1134. 10.1016/0091-6749(89)90456-9View ArticlePubMedGoogle Scholar
- Smith P, Suphioglu C, Griffith IJ, Theriault K, Knox RB, Singh MB: Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen. J Allergy Clin Immunol. 1996, 98: 331-343. 10.1016/S0091-6749(96)70158-6View ArticlePubMedGoogle Scholar
- Chang ZN, Peng HJ, Lee WC, Chen TS, Chua KY, Tsai LC, Chi CW, Han SH: Sequence polymorphism of the group 1 allergen of Bermuda grass pollen. Clin Exp Allergy. 1999, 29: 488-496. 10.1046/j.1365-2222.1999.00523.xView ArticlePubMedGoogle Scholar
- Au L, Lin ST, Peng HJ, Liang CC, Lee SS, Liao CD, Chang ZN: Molecular cloning and sequence analysis of full-length cDNAs encoding new group of Cyn d 1 isoallergens. Allergy. 2002, 57: 215-220. 10.1034/j.1398-9995.2002.1o3162.xView ArticlePubMedGoogle Scholar
- Leifermann K, Gleich GJ: The cross reactivities of IgE antibodies with pollen antigens. Analysis of various species of grass pollens. J Allergy Clin Immunol. 1976, 158: 129-139. 10.1016/0091-6749(76)90148-2.View ArticleGoogle Scholar
- Chakrabarty S, Loewenstein H, Ekramoddullah AK, Kisil FT, Schon AH: Detection of cross-reactivite allergens in Kentucky blue grass pollen and six other grasses by crossed radioimmunoelectrophoresis. Int Arch Allergy Appl Immunol. 1981, 66: 142-147.View ArticlePubMedGoogle Scholar
- Davies JM, Bright ML, Rolland JM, O'Hehir RE: Bahia grass pollen specific IgE is common in seasonal rhinitis patients but has limited cross-reactivity with Ryegrass. Allergy. 2005, 60: 251-255. 10.1111/j.1398-9995.2005.00663.xView ArticlePubMedGoogle Scholar
- Martin B, Mansfield LE, Nelson HS: Cross-allergenicity among the grasses. Ann Allergy. 1985, 54: 99-104.PubMedGoogle Scholar
- Schumacher M, Grabowski J, Wagner CM: Anti-Bermuda grass RAST binding is minimally inhibited by pollen extracts from ten other grasses. Ann Allergy. 1985, 55: 584-587.PubMedGoogle Scholar
- Duffort O, Quintana J, Ipsen H, Barber D, Polo F: Antigenic Similarity among Group 1 Allergens from Grasses and Quantitation ELISA Using Monoclonal Antibodies to Phl p 1. Int Arch Allergy Immunol. 2008, 145: 283-290. 10.1159/000110887View ArticlePubMedGoogle Scholar
- Lamontagne P, Boutin Y, Brunet C, Boulanger J, Berton J, Hebert J: Characterization of allergenic determinants on the C-terminal region of the r-Lol p 1. Adv Exp Med Biol. 1996, 409: 425-429.View ArticlePubMedGoogle Scholar
- Schramm G, Bufe A, Petersen A, Haas H, Schlaak M, Wolf-Meinhard B: Mapping of IgE-binding epitopes on the recombinant major group 1 allergen of velvet grass pollen, rHol l 1. J Allergy Clin Immunol. 1997, 99: 781-787. 10.1016/S0091-6749(97)80012-7View ArticlePubMedGoogle Scholar
- Ball T, Fuchs T, Sperr WR, Valent P, Vangelista L, Kraft D, Valenta R: B cell epitopes of the major timothy grass pollen allergen, Phl p 1, revealed by gene fragmentation as candidates for immunotherapy. The FASEB journal. 1999, 13: 1277-1290.PubMedGoogle Scholar
- Logemann J, Schell J, Willmitzer L: Improved method for the isolation of RNA from plant tissues. Anal Biochem. 1987, 163: 16-20. 10.1016/0003-2697(87)90086-8View ArticlePubMedGoogle Scholar
- Esch R, Klapper DG: Isolation and characterization of a major cross-reactive grass group 1 allergenic determinant. Mol Immunol. 1989, 26: 557-561. 10.1016/0161-5890(89)90007-2View ArticlePubMedGoogle Scholar
- Tamborini E, Faccini S, Lidholm J, Svensson M, Brandazza A, Longhi R, Groenlund H, Sidoli A, Arosio P: Biochemical and immunological characterization of recombinant allergen Lol p 1. Eur J Biochem. 1997, 249: 886-894. 10.1111/j.1432-1033.1997.00886.xView ArticlePubMedGoogle Scholar
- Linhart B, Valenta R: Vaccine engineering improved by hybrid technology. Int Arch Allergy Immunol. 2004, 134: 324-331. 10.1159/000079535View ArticlePubMedGoogle Scholar
- Flicker S, Steinberger P, Ball T, Krauth MT, Verdino P, Valent P, Almo S, Valenta R: Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1:Importance for allergenic activity. J Allergy Clin Immunol. 2006, 117: 1336-13431. 10.1016/j.jaci.2006.02.012View ArticlePubMedGoogle Scholar
- de Weerd N, Bhalla PL, Singh MB: Effect of cysteine mutagenesis on human IgE reactivity of recombinants forms of the major rye grass pollen allergen Lol p 1. Allergol Int. 2003, 52: 183-190. 10.1046/j.1323-8930.2003.00300.x.View ArticleGoogle Scholar
- Breiteneder H, Ferreira F, Hoffmann SK, Ebner C, Breitenbach M, Rumpold H, Kraft D, Scheiner O: Four recombinant isoforms of Cor a 1, the major allergen of hazel pollen, show different IgE-binding properties. Eur J Biochem. 1993, 212: 355-362. 10.1111/j.1432-1033.1993.tb17669.xView ArticlePubMedGoogle Scholar
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