NOVEL ALLERGEN
20170219602 · 2017-08-03
Assignee
Inventors
- Lars Mattsson (Uppsala, SE)
- Ulrica OLSSON (Uppsala, SE)
- Thomas Lundgren (Uppsala, SE)
- Jonas Lidholm (Knivsta, SE)
- Håkan LARSSON (Bälinge, SE)
Cpc classification
International classification
Abstract
The invention relates to a novel horse allergen consisting of a heterodimeric protein having a first peptide chain and a second peptide chain together having an overall sequence identity of at least 70% with the combined sequences of SEQ ID NO:3 and SEQ ID NO: 4, as well as a single chain protein having an overall sequence identity of at least 70%, such as 75%, 80%, 85%, 90%, 95%, or 98%, with the combined amino acid sequences according to SEQ ID NO: 3 and SEQ ID NO: 4. The invention further relates to the use of the protein in methods of diagnosis and therapy of Type I allergy, and kits and compositions for use in such methods.
Claims
1.-27. (canceled)
28. An isolated heterodimeric protein having a first peptide chain and a second peptide chain together having an overall sequence identity of at least 70% with the combined sequences of SEQ ID NO: 3 and SEQ ID NO: 4.
29. The isolated heterodimeric protein according to claim 28, having an overall sequence identity of at least 75%, more preferably 80%, 85%, 90%, 95%, or 98%, with the combined sequences of SEQ ID NO: 3 and SEQ ID NO: 4.
30. A single chain protein having an overall sequence identity of at least 70%, such as 75%, 80%, 85%, 90%, 95%, or 98%, with the combined amino acid sequences according to SEQ ID NO: 3 and SEQ ID NO: 4.
31. An isolated protein having a sequence identity of at least 70%, such as 75%, 80%, 85%, 90%, 95%, or 98%, with the sequence of SEQ ID NO: 3.
32. An isolated protein having a sequence identity of at least 70%, such as 75%, 80%, 85%, 90%, 95%, or 98%, with the sequence of SEQ ID NO: 4.
33. A fragment of a protein according to claim 28, comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4.
34. A protein according to claim 28, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4, which has been immobilized to a solid or soluble support and/or which has been provided with a detectable label.
35. A nucleic acid molecule coding for a protein according to claim 28, or for a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4.
36. A vector comprising the nucleic acid molecule according to claim 35.
37. A host cell comprising the vector according to claim 36.
38. A method for in vitro assessment of type 1 allergy comprising the steps of contacting an immunoglobulin-containing body fluid sample from a patient suspected of having Type 1 allergy with a protein according to claim 28, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4; and detecting the presence, in the sample, of antibodies specifically binding to said protein or protein fragment; wherein the presence of such bound antibodies is indicative of a Type 1 allergy in said patient.
39. A method according to claim 38, which comprises detecting the presence, in the sample, of IgE and/or IgG antibodies specifically binding to said protein or protein fragment, wherein the presence of specific IgE antibodies is indicative of a Type 1 allergy to horse in said patient and the level of specific IgG antibodies is informative in regard to natural or induced tolerance to horse through environmental exposure or immunotherapy treatment.
40. The method according to claim 38, further comprising the steps of contacting the immunoglobulin-containing body fluid sample from the patient suspected of having Type 1 allergy with at least one further purified allergen component from horse; and detecting the presence, in the sample, of IgE antibodies specifically binding to said purified allergen component from horse; wherein the combination of presence of IgE antibodies specifically binding to said protein or protein fragment, and absence of IgE antibodies specifically binding to said allergen component from horse, is indicative of a Type 1 allergy to cat in said patient.
41. The method according to claim 40, wherein the further purified allergen component from horse is selected from the group consisting of native and recombinant Equ c 1, Equ c 2, Equ c 3, Equ c 4/5, and Equ c 15k.
42. A kit for performing the method according to claim 38, comprising the protein or protein fragment immobilised on a solid support.
43. The single chain protein of claim 30, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4, which has been immobilized to a solid or soluble support and/or which has been provided with a detectable label.
44. A method for in vitro assessment of type 1 allergy comprising the steps of contacting an immunoglobulin-containing body fluid sample from a patient suspected of having Type 1 allergy with the single chain protein according to claim 30, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4; and detecting the presence, in the sample, of antibodies specifically binding to said protein or protein fragment; wherein the presence of such bound antibodies is indicative of a Type 1 allergy in said patient.
45. The isolated protein of claim 31, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4, which has been immobilized to a solid or soluble support and/or which has been provided with a detectable label.
46. The isolated protein of claim 32, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4, which has been immobilized to a solid or soluble support and/or which has been provided with a detectable label.
47. A method for in vitro assessment of type 1 allergy comprising the steps of contacting an immunoglobulin-containing body fluid sample from a patient suspected of having Type 1 allergy with the isolated protein according to claim 31, or a fragment thereof comprising at least one IgE antibody epitope of a heterodimeric protein having a first peptide chain having the sequence according to SEQ ID NO: 3 and a second peptide chain having the sequence according to SEQ ID NO: 4; and detecting the presence, in the sample, of antibodies specifically binding to said protein or protein fragment; wherein the presence of such bound antibodies is indicative of a Type 1 allergy in said patient.
Description
BRIEF DESCRIPTION OF DRAWINGS
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SEQUENCE LISTING
[0060] The following sequences are listed in the sequence listing.
TABLE-US-00001 SEQ ID NO: Description 1 amino acid sequence of the 5 kDa chain of Equ c s including signal peptide 2 predicted amino acid sequence of the 10 kDa chain of Equ c s including signal peptide 3 predicted amino acid sequence of the 5 kDa chain of Equ c s 4 predicted amino acid sequence of the 10 kDa chain of Equ c s 5 amino acid sequence for the whole recombinant protein rEqu c s ab 6 nucleic acid sequence encoding the whole recombinant protein rEqu c s ab 7 amino acid sequence for the alternative recombinant protein rEqu c s ba 8 nucleic acid sequence encoding the alternative recombinant protein rEqu c s ba 9 Codon optimized nucleic acid molecule encoding chain 1 10 Codon optimized nucleic acid molecule encoding chain 2 11 Forward primer for chain 1, PCR 1 12 Forward primer for chain 2, PCR 1 13 Forward primer for chain 1, PCR 2 14 Forward primer for chain 2, PCR 2 15 Reverse primer for chain 1, PCR 2 16 Reverse primer for chain 2, PCR 2 17-33 Peptide fragments disclosed in Table 7 34-43 Peptide fragments disclosed in Table 8 44 Double sequence given by N-terminal sequencing analysis by Edman degradation of the RPC peak 2 45 The complete DNA sequence of the postulated sequence denoted Equ c s, chain 1 46 The complete DNA sequence of the postulated sequence denoted Equ c s, chain 2 47 Fel d 1 chain 1 48 Fel d 1 chain 2
DETAILED DESCRIPTION OF THE INVENTION
[0061] In one aspect the present invention relates to an isolated horse allergen, herein denoted Equ c s, belonging to the secretoglobin family, showing an electrophoretic mobility (apparent molecular weight) corresponding to approximately 18 kDa under non-reducing conditions, and comprising a first peptide chain having a molecular weight in the order of 5 kDa and a second peptide chain having a molecular weight in the order of 10 kDa, linked together by one or more disulphide bonds. This aspect of the invention also comprises variants and fragments of Equ c s with certain sequence identity to native Equ c s, as defined above, and preferably comprising at least one IgE antibody epitope of native Equ c s. Such variants and fragments preferably have IgE reactivity to Equ c s reactive sera and at least 10% of the IgE binding to the original rEqu c s molecule can be inhibited by such a variant or fragment, as determined by the assay described in Example 10. Also in the other aspects of the invention described below, the term “Equ c s” is, for simplicity, used to also include such variants and fragments thereof.
[0062] In another aspect, the invention relates to an isolated nucleic acid molecule encoding the allergen according to the first-mentioned aspect, as well as to a vector containing the nucleic acid molecule, and to a host cell containing the vector. Recombinant proteins or peptides produced by such a vector-containing host cell may be glycosylated or not depending on the host cell used.
[0063] In a further aspect, the invention relates to an in vitro method for assessment of a Type I allergy in a patient, wherein a body fluid sample, such as a blood or serum sample from the patient, is brought into contact with Equ c s or a composition according to the previous aspect, whereby it can be determined whether or not the patient sample contains IgE antibodies that bind specifically to the Equ c s. Such a method may be carried out in any manner known in the art. The Equ c s may e.g. be immobilized on a solid support, such as in a conventional laboratory immunoassay, in a microarray or in a lateral flow assay, or used as a fluid-phase reagent.
[0064] In yet a further aspect, the invention relates to an in vitro method for assessment of a Type I allergy in a patient, wherein a body fluid sample, such as a blood or serum sample from the patient, is brought into contact with Equ c s, whereby it can be determined whether or not the patient sample contains IgE antibodies that bind specifically to the Equ c s but not to other horse allergen components, such as Equ c 1, Equ c 2, Equ c 3, Equ c 4/5 or Equ c 15k. A patient showing IgE reactivity against Equ c s, but not against other horse allergen components, is likely to be primarily sensitised to cats and not to horses.
[0065] In the above mentioned aspects, the wildtype Equ c s molecule may, as mentioned above, be replaced with fragments or variants of Equ c s, natural or man-made, comprising IgE antibody epitopes from the wildtype protein.
[0066] The invention further relates to a method of treatment of Type I allergy comprising administering to a patient in need of such treatment Equ c s or a modified Equ c s, as explained below. This aspect of the invention also relates to the use of the Equ c s in such immunotherapy, including e.g. component-resolved immunotherapy (Valenta and Niederberger 2007). In one embodiment of this aspect, the Equ c s may be used in its natural form or in a recombinant form displaying biochemical and immunological properties similar to those of the natural molecule. In another embodiment, the Equ c s may be used in a modified form, generated chemically or genetically, in order to abrogate or attenuate its IgE antibody binding capacity, while preferably being capable of eliciting an IgG response in a treated individual. Examples of modifications include, but are not limited to, fragmentation, truncation, tandemerization or aggregation of the molecule, deletion of internal segment(s), substitution of amino acid residue(s), domain rearrangement, or disruption at least in part of the tertiary structure by disruption of disulfide bridges or its binding to another macromolecular structure, or other low molecular weight compounds. In yet another embodiment of this aspect, the individual 10 kDa and/or 5 kDa subunits of Equ c s are used as modified Equ c s.
[0067] In all of the above mentioned aspects of the invention, the Equ c s protein may be purified from its natural source, such as from urine, saliva or other body fluids, or from tissue, such as hair or dander, from horse. It may also, as mentioned above, be produced by recombinant DNA technology or chemically synthesized by methods known to a person skilled in the art or described in the present application.
[0068] The allergenic horse protein described here, Equ c s, belongs to the secretoglobin protein family, specifically one subfamily which comprises tetrameric proteins formed by two heterodimeric subunits. The heterodimer consists of two chains derived from different genes linked together by disulfide bridges (Klug et al. 2000). The horse secretoglobin described here is a 18±2 kDa heterodimer, herein referred to as Equ c s, consisting of a 5±2 kDa and a 10±2 kDa subunit, respectively, which for the purposes of this invention are referred to as the 5 and 10 kDa subunits, respectively. The molecular weight assignments are according to their apparent molecular weight as observed in SDS-PAGE, as described in Example 3 below. It is understood that the apparent molecular weights will vary depending on the separation conditions, including electrophoretic separation medium and concentration thereof, linear or gradient buffer used, etc. Also, the 10 kDa subunit contains an N-glycosylation site, the occupation of which by a glycan structure may affect the apparent molecular weight.
[0069] The amino acid sequence of the 5 kDa chain has the predicted amino acid sequence
[0070] DICPAVKEDV NIFLTGTPDD YVKKVSQYQR NPVILANAEK LKNCIDKKLT AEDKENALSV 60
[0071] LEKIYSSDFC 70
[0072] (SEQ ID NO: 3) and a theoretical molecular weight of 7.9 kDa.
[0073] The amino acid sequence of the 10 kDa chain has the predicted amino acid sequence
[0074] CPSFYAVLGV LSLGSKTLLD TSLNLVNATE PEKVAMGKIQ DCYNEAGVIT KISDLIIMGT 60
[0075] ITTSPECISH ALSTLTTDVQ EGISKLNPLG R 91
[0076] (SEQ ID NO: 4) and a theoretical molecular weight of 9.6 kDa.
[0077] It is to be noted that structurally related proteins have been described in a wide range of mammalian species but only two proteins have been defined as allergens, the major cat allergen Fel d 1 (Acc no P30438 and P30440) and Equ c 15k (WO2011/133105).
[0078] Although the horse dander allergens Equ c 1, Equ c 2, Equ c 3, Equ c 4/5 and Equ c 15k cover most of the IgE reactivity to horse dander extract observed in horse allergic patients, we have encountered several cases of cat allergic individuals demonstrating IgE reactivity to horse dander extract without concomitant reactivity to any of the five known horse allergen components. This invention describes the identification and characterisation of the horse allergen responsible for this unknown IgE reactivity to horse dander extract leading to the discovery of a protein homologue to the cat allergen Fel d 1.
[0079] In a collection of sera from cat sensitised subjects a number of sera could be characterised having reactivity to horse dander extract while no reactivity to any of the known horse dander allergens could be detected. Using the sera described above, the IgE binding to horse dander extract could be inhibited by recombinant Fel d 1, indicating that the IgE reactivity is directed to a horse protein that is immunologically similar to Fel d 1.
[0080] With the aid of these sera, a new major allergen could be purified from horse dander and identified as a member of the secretoglobin protein family. The novel horse protein, herein referred to as Equ c s, consists of one 5 kDa amino acid chain and one 10/11 kDa amino acid chain joined together by disulfide bridges. Considering the fact that the two polypeptide chains are encoded by separate genes, this study demonstrates the presence of a heterodimeric protein that has not previously been anticipated by bioinformatic studies of the horse genome. It is distinct from previously known horse allergens. This allergen represents an important addition to the panel of known horse allergens and will be useful in the diagnosis of horse allergy. Since this is an allergen that is cross reactive to the main cat allergen Fel d 1, IgE reactivity to this molecule may reflect cross reactive sensitisation to horse dander that may or may not be associated to clinical symptoms.
[0081] The examples below illustrate the present invention with the isolation and use of the secretoglobin denoted Equ c s from horse. The examples are only illustrative and should not be considered as limiting the invention, which is defined by the scope of the appended claims.
Example 1: Identification of Sera Detecting an Unknown Allergen Component in Horse Dander Extract that is Similar to Fel d 1
[0082] Horse dander extract, cat dander extract and Fel d 1 regular ImmunoCAP tests were used. Experimental immunoCAP tests using recombinant Equ c 1 and Equ c 15k as well as Equ c 2 and Equ c 4 purified from horse dander and Equ c 3 purified from horse serum were produced essentially as described in Patent WO2011/133105).
[0083] A collection of sera having high level of sensitisation to cat dander extract were tested for IgE reactivity to cat and horse dander components. The five sera identified here were characterised by high IgE reactivity to cat dander extract, Fel d 1 and horse dander extract without concomitant reactivity to any of the five known horse allergen components (table 1). These sera thus detected an unknown allergen component in horse dander extract.
[0084] The selected sera were utilised in inhibition tests using both horse dander extract and cat dander extract as solid phase (table 2). As inhibitors recombinant Equ c 15k, Fel d 1 and Fel d 7 respectively were used at a final concentration of 100 μg/ml. As an inhibition control buffer, 0.1 M sodium phosphate buffer, pH 7.4, containing 0.3% human serum albumin was used. Means of duplicate determinations of each inhibition were calculated and the fraction of inhibition was calculated as the fraction of the binding using inhibition control buffer that could be quenched with each inhibitor. In these selected sera, binding to cat dander extract could be almost completely inhibited by Fel d 1 (table 2a). None of the other inhibitors tested showed any inhibition. This demonstrates that the IgE binding to cat dander extract is dominated by reactivity to Fel d 1. Likewise, the binding to horse dander extract solid phase (table 2b) could be fully inhibited by rFel d 1 but none of the other inhibitors. This demonstrated that the binding to horse dander extract by these sera is directed to a horse dander component that is immunologically similar to the cat allergen Fel d 1. Although Equ c 15k belongs to the secretoglobin family this protein did not demonstrate any inhibition of the binding to horse dander extract, indicating that the horse component searched for is not Equ c 15k. This can be explained by the fact that the two proteins belong to different sectretoglobin subfamilies, Fel d 1 belongs to the B-E subfamily and Equ c 15k belongs to the C-D subfamily (Laukaitis and Karn 2005), WO2011/133105
Example 2: Purification of a Horse Dander Allergen Component, Homologous to the Cat Allergen Fel d 1
[0085] By use of the sera described in example 1, an unknown allergen component, similar to Fel d 1, could be detected in horse dander extract and by fractioning horse dander extract by chromatographic procedures and immobilising these fractions on an ImmunoCAP solid phase, the unknown component could be followed during several chromatographic steps.
Size Exclusion Chromatography
[0086] Horse dander (Allergon, Välinge, Sweden) was extracted in 20 mM MOPS, pH 7.6, 0.15 M NaCl (MBS=MOPS-buffered saline), clarified by centrifugation and filtered through a 0.45 μm mixed cellulose ester filter (Millipore, Billerica, Mass., USA). As a first purification step, the clarified extract was applied to a Superdex™ 75 column (XK26/100, V.sub.t=505 mL, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) for size exclusion chromatography (SEC) and elution was performed with MBS at a flow rate of 2 mL/min.
[0087] The chromatogram is shown in
Hydrophobic Interaction Chromatography
[0088] The pool from SEC was adjusted to 1 M NH.sub.4SO.sub.4 and applied to a Phenyl Sepharose™ HP column (HR10/10, V.sub.t=8.0 mL, GE Healthcare Life Sciences) equilibrated with 1 M NH.sub.4SO.sub.4 in 20 mM tris pH 8.0. Elution was performed in a linear NH.sub.4SO.sub.4 gradient from 1 M to 0 M NH.sub.4SO.sub.4 (indicated as 50%-100% B in the chromatogram in
Anion Exchange Chromatography
[0089] The HIC pool was conditioned by adding half the volume of the pool of Tris pH 8.5 to the HIC pool. The pool was subsequently applied to an anion exchange column Source™ 15Q (PE4.6/100, V.sub.t=1.66 mL, GE Healthcare Life Sciences) equilibrated with 20 mM Tris, pH 8.5. Upon elution in a linear 0-0.50 M NaCl gradient in the same buffer the protein was resolved into several peaks (
Reversed-Phase Chromatography
[0090] The anion exchange pool was conditioned by adding TFA to a final concentration of 0.065% and subjected to a final RPC purification step by applying the sample to a Source™ 15 RPC column (Resource, V.sub.t=3.2 mL; GE Healthcare Life Sciences) equilibrated with 0.065% TFA in water. Elution was performed in a linear 0-60% gradient of buffer B, consisting of 0.05% TFA in 90% acetonitrile. A Three peaks were eluted near the end of the gradient (
Example 3: Analysis of the Purified Fraction by SDS PAGE, N-Terminal Sequencing and MALDI TOF MS
[0091] SDS PAGE analysis of the RPC fractions revealed a similar pattern for the first two RPC peaks, one 5 kDa band and a double band at 10/11 kDa under reducing conditions that came together at a broad band at 18 kDa under non-reducing conditions. This band pattern is consistent with proteins from the secretoglobin family that typically contain two bands at 5 and 10 kDa under reducing conditions and one band at 15-20 kDa under non-reducing conditions. The largest of the two bands band is glycosylated and therefore may appear diffuse or as in this case appear as a double band.
[0092] N-terminal sequencing analysis by Edman degradation of the RPC peak 2, performed essentiallly as described in (Mattsson, Lundgren et al. 2009), revealed a double sequence that became less clear after seven residues:
Amino acid residue no: 1 2 3 4 5 6 7
First alternative D P S F Y A V
Second alternative - I R P A V -
[0093] Since the relative amounts of the amino acids in each cycle were similar it was not possible to establish a primary and a secondary sequence from these data. The double sequence is also shown in the sequence listing as in SEQ ID NO: 44.
[0094] In gel digestion with trypsin on spots from one dimensional SDS PAGE gel electrophoretic bands 5, 10 and 11 kDa followed by analysis by matrix assisted laser desorption ionization time of flight mass-spectrometry (MALDI TOF MS) using a Bruker Daltonics Autoflex 2 instrument (Bruker Daltonics, Bremen, Germany) followed by peptide mass fingerprint (PMF) analysis did not result in significant matches with any known proteins in the NCBI-NR database except for the 5 kDa band that matched a predicted sequence of an uncharacterised horse protein (XP_005596696). However, although a part of the sequence of this record was homologous to secretoglobins, the molecular weight was too high for this family of molecules and did not match the band that it was picked out from. It was assumed that this predicted record was faulty.
Example 4: Bioinformatic Analysis of Horse Genomic Sequences Identifying Amino Acid Sequences Homologous to Chain 1 and Chain 2 of Fel d 1
Chain 1
[0095] The cat allergen protein Fel d 1, which was immunologically similar to the unknown horse dander protein as demonstrated in example 1, consists of two amino acid chains, chain 1 and chain 2 (acc no: NP_001041618 and NP_001041619 respectively) joined together by disulfide bridges.
[0096] A TBLASTN search of a horse genome database (wsg) with the sequence of Fel d 1, chain 1 (NP_001041618) resulted in a match of aa 17-79 to the translation of nucleotide positions 77633-77453 (of the reverse strand) of Acc No. AAWR02030062, a 105199 bp segment of the horse genome sequence.
[0097] A larger segment surrounding this sequence, 90000-70021 of Acc No. AAWR02030062 was fed into the gene finding program FGENESH+ together with the precursor sequence of Fel d 1 chain 1. The program searches for homologous genes within a genomic sequence.
[0098] The result was a postulated sequence consisting of three exons Acc No. AAWR02030062:
77850->77790
77632->77445
76428->76399
[0099] The complete DNA sequence of this postulated sequence, denoted Equ c s, chain 1, is shown in
Chain 2
[0100] Similarly to above A TBLASTN search of a horse genome database (wsg) with the sequence of Fel d 1, chain 2 (NP_001041619) resulted in a match of aa 21-85 to the translation of nucleotide positions 82588-82782 (of the forward strand) of Acc No. AAWR02030062, a 105199 bp segment of the horse genome sequence.
[0101] A larger segment surrounding this sequence, 70021-94020 of Acc No. AAWR02030062 was fed into the gene finding program FGENESH+ together with the precursor sequence of Fel d 1 chain 2. The program searches for homologous genes within a genomic sequence.
[0102] The result was an incomplete postulated sequence consisting of two exons from Acc No. AAWR02030062:
82004->82064
82589->82770
[0103] Based on homology with the sequence for Fel d 1 chain 2, the last exon is missing in this postulated sequence. A protein BLAST search comparing exon 3 of Fel d 1 chain 2 with the translated genomic sequence following nukleotide 82770 identified a genomic sequence having homology to exon 3 of Feld 1 chain 2. This postulated exon 3 of Equ c s could be joined in frame with the previous exons and contained a stop codon at an homologous position to exon 3 of Fel d 1 chain 2. The sequence of this final exon was found at: 90025-90127 of the genomic sequence Acc No. AAWR02030062.
[0104] The complete DNA sequence of this postulated sequence, denoted Equ c s, chain 2, is shown in
Example 5: PCR Amplification and Sequencing of Equ c s Chain 1 and Chain 2 mRNA from Horse Skin
[0105] Total RNA was prepared from horse skin using the RNAqueous kit (Ambion, Austin, Tex., USA). Polyadenylated RNA was isolated from total RNA using the mRNA Purification kit and first strand cDNA was prepared using the First-Strand cDNA Synthesis kit (both from Thermo Fisher Scientific). 3′ RACE was performed according to Frohman (Frohman 1993), using a gene-specific forward oligonucleotide primer from the untranslated sequence before the starting codon,
TABLE-US-00002 (SEQ ID NO: 11, chain 1) 5′-ATAAAAGGGCTGCAGAATTG-3′ and (SEQ ID NO: 12, chain 2) 5′-GCAGCAGAAACCCTGCCCTG-3′.
[0106] A second PCR was performed using a second gene-specific forward oligonucleotide primer from the untranslated sequence before the starting codon,
TABLE-US-00003 (SEQ ID NO: 13, chain 1) 5′-GTGAGCACCTGCCACCTG-3′ and (SEQ ID NO: 14, chain 2) 5′-GAAGAGCATTCTAGCAGTTG-3′
[0107] carrying a terminal NdeI restriction site for cloning and specific reverse oligonucleotide primers,
TABLE-US-00004 (SEQ ID NO: 15, chain 1) 5′-GAATCTTCTAATCAGACAC-3′ and (SEQ ID NO: 16, chain 2) 5′-GGTAGAGGAGACAGGTGTC-3′.
[0108] Four independent 3′ RACE clones for chain 1 and three independent 3′ RACE clones for chain 2 were isolated and sequenced in their entirety whereby the coding sequence of the postulated chains of Equ c s could be verified. DNA sequencing was performed using an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA). DNA and amino acid sequence analyses and calculations were performed using programs of the GCG Wisconsin Package (Accelrys, San Diego, Calif., USA).
Example 6: N-Terminal Sequencing and MALDI TOF Analysis Using the Postulated Sequences of Equ c s Chain 1 and Chain 2
[0109] Re-evaluation of the double amino acid sequence described in example 3 was performed using the postulated sequences of Equ c s. The double sequence can now be interpreted as DICPAV (residues 1-6 of SEQ ID NO: 3) and CPSFYAV (residues 1-7 of SEQ ID NO: 4) which are identical to the postulated N-terminal sequences of the mature chain 1 and chain 2 of the Equ c s protein.
[0110] In gel digestion of the 5, 10 and 11 kDa bands of the reduced sample of peak 2 in
[0111] In the 11 kDa band two different peptides were detected that matched the mass of hypothetical trypsin cleaved peptides in the postulated Equ c s chain 2 sequence (table 7b). These peptides covered 32% of the postulated mature sequence including the N-terminal end of the amino acid chain. The reason that only a small part of the chain 2 sequence could be identified is that it contains an N-glycosylation and the fact that the trypsin cleavage sites are distributed so that either too large or too small peptides are produced. In the 18 kDa band, peptides from both chain 1 and chain 2 could be found.
[0112] In order to increase the signal strength of these peptides to enable MS/MS analysis, two fractions from an RPC chromatography purification similar to that as described in example 2 but using a steeper gradient (
[0113] The SDS-PAGE analysis (
Example 7: Production and Immunological Characterization Recombinant Equ c s Cloning and Purification of Recombinant Equ c s
[0114] A synthetic Equ c s single chain gene was designed by combining nucleotide sequences encoding the amino acid sequences of the 5 kDa and the 10 kDa subunits with a sequence encoding a linker peptide comprising 3× (Gly-Gly-Gly-Gly-Ser, residues 72-86 of SEQ ID NO: 5). The full-length synthetic gene was cloned into the NdeI and XhoI sites of vector pET23a(+) (Novagen, Madison, Wis., USA), adding a C-terminal hexahistidine tag to enable protein purification by immobilised metal ion affinity chromatography (IMAC).
[0115] The amino acid sequence for the whole recombinant protein (denominated rEqu c s ab) is
TABLE-US-00005 (SEQ ID NO: 5) MDICPAVKED VNIFLTGTPD DYVKKVSQYQ RNPVILANAE KLKNCIDKKL TAEDKENALS 60 VLEKIYSSDF CGGGGSGGGG SGGGGSCPSF YAVLGVLSLG SKTLLDTSLN LVNATEPEKV 120 AMGKIQDCYN EAGVITKISD LIIMGTITTS PECISHALST LTTDVQEGIS KLNPLGRLEH 180 HHHHH. 185
[0116] The nucleotide sequence was designed for optimal codon usage in E. coli (DNA2.0, Menlo Park, Calif., USA).
[0117] The nucleic acid sequence encoding the whole recombinant protein is
TABLE-US-00006 (SEQ ID NO: 6) atggacattt gccctgcggt taaagaggac gtcaacattt ttctgaccgg taccccagat 60 gattacgtca aaaaagtgag ccagtaccag cgtaacccgg ttattctggc aaatgccgag 120 aaactgaaga attgtatcga caaaaagctg acggctgagg ataaggaaaa cgccctgtct 180 gtcttggaga agatttacag cagcgacttc tgtggtggcg gtggcagcgg tggtggtggt 240 tcgggcggtg gcggcagctg cccgtccttc tatgcggtgc tgggtgttct gagcttaggt 300 agcaagaccc tgttggacac gagcctgaat ttggtgaatg cgactgaacc ggagaaagtc 360 gcaatgggca agatccaaga ttgctataac gaagcgggcg ttatcaccaa gatcagcgat 420 ctgatcatta tgggtacgat cacgaccagc ccggaatgta tctctcacgc gctgtccacc 480 ctgaccaccg acgtgcaaga gggcattagc aaactgaacc cgctgggtcg cctcgagcac 540 caccaccacc accac. 555
[0118] The nucleic acid sequence encoding chain 1 is
TABLE-US-00007 (SEQ ID NO: 9) gacatttgcc ctgcggttaa agaggacgtc aacatttttc tgaccggtac cccagatgat 60 tacgtcaaaa aagtgagcca gtaccagcgt aacccggtta ttctggcaaa tgccgagaaa 120 ctgaagaatt gtatcgacaa aaagctgacg gctgaggata aggaaaacgc cctgtctgtc 180 ttggagaaga tttacagcag cgacttctgt 210
[0119] The nucleic acid encoding chain 2 is
TABLE-US-00008 (SEQ ID NO: 10) tgcccgtcgt tttatgcagt cctgggtgtt ctgtctttgg gttctaaaac tttgctggac 60 acgagcctga atctggtgaa tgcaacggag cctgaaaagg tcgcgatggg caagattcag 120 gactgttaca acgaagcggg cgttattacc aagatcagcg acctgatcat tatgggcacg 180 atcaccacga gcccagagtg catcagccac gctttgtcca ccctgaccac cgatgtccaa 240 gagggcatta gcaagctgaa cccgctgggt cgc 273
[0120] An alternative construct was designed having the 10 kDa subunit at the N-terminal end followed by the linker and the 5 kDa subunit, thereby connecting the two subunits in the other end of each amino acid chain. The amino acid sequence for the alternative recombinant protein (denominated rEqu c s ba) is
TABLE-US-00009 (SEQ ID NO: 7) MCPSFYAVLG VLSLGSKTLL DTSLNLVNAT EPEKVAMGKI QDCYNEAGVI TKISDLIIMG 60 TITTSPECIS HALSTLTTDV QEGISKLNPL GRGGGGSGGG GSGGGGSDIC PAVKEDVNIF 120 LTGTPDDYVK KVSQYQRNPV ILANAEKLKN CIDKKLTAED KENALSVLEK IYSSDFCLEH 180 HHHHH 185
and the nucleic acid sequence encoding the same protein is
TABLE-US-00010 (SEQ ID NO: 8) atgtgcccgt cgttttatgc agtcctgggt gttctgtctt tgggttctaa aactttgctg 60 gacacgagcc tgaatctggt gaatgcaacg gagcctgaaa aggtcgcgat gggcaagatt 120 caggactgtt acaacgaagc gggcgttatt accaagatca gcgacctgat cattatgggc 180 acgatcacca cgagcccaga gtgcatcagc cacgctttgt ccaccctgac caccgatgtc 240 caagagggca ttagcaagct gaacccgctg ggtcgcggtg gtggcggtag cggtggtggt 300 ggctccggtg gcggtggcag cgatatttgt ccggcggtga aagaagatgt caacatcttc 360 ctgaccggta ccccggatga ttatgtgaaa aaagttagcc aataccagcg taatccggtt 420 atcctggcca atgccgagaa actgaagaac tgcatcgaca aaaagctgac cgcagaggac 480 aaagaaaacg cgctgagcgt gctggagaag atttacagca gcgacttctg tctcgagcac 540 caccaccacc accac. 555
[0121] The plasmid DNA constructs was transformed into E. coli strain BL21-AI (Invitrogen) and recombinant Equ c s single chain protein was produced using a 3-litre bioreactor (Belach Bioteknik, Skogås, Sweden).
[0122] The method of purification of recombinant Equ c s were almost identical for the two variants of the protein. Harvested cells was resuspended in 20 mM Tris-HCl pH 8.0 and lysed by passing the suspension through an Emulsiflex C5 homogenizor (Avestin, Ottawa, Ontario, Canada) at 10 000-15 000 kPa. After clarification by centrifugation and filtration, the supernatant was applied to a Chelating Sepharose FF column (GE Healthcare Life Sciences), charged with NiSO.sub.4. Column washing was performed with 20 mM imidazole in 20 mM Tris-HCl pH 8.0, 0.15 M NaCl and the recombinant protein eluted in a linear 20-500 mM gradient of imidazole in the same buffer (
Biochemical Characterisation of Recombinant Equ c s
[0123] Analytical gel filtration of peak 1 and peak 2 of rEqu c s ab demonstrated that peak 1 contained a mixture of dimeric and monomeric form of rEqu c s (
[0124] SDS PAGE analysis of single chain recombinant Equ c s demonstrated a single band at 19 kDa for reducing conditions and a slightly lower apparent molecular weight band at 16 kDa for nonreducing conditions (
[0125] N-terminal sequence analysis of the first variant of rEqu c s (rEqu c s ab) resulted in a clear and unambiguous sequence readout with no deviation from expected sequence and where the initiator methionine was fully retained. In the preparation of the second variant, rEqu c s ba, the initiator methionine was retained in approximately half of the sample but the remainder of the preparation started at the second amino acid chain. In conclusion, both of the recombinant preparations contained intact rEqu c s.
Assessment of IgE Binding to Recombinant Equ c s
[0126] Each of peak 1 and 2 of the two forms of recombinant Equ c s was immobilised to experimental ImmunoCAP™ as described (Marknell DeWitt, Niederberger et al. 2002) and the IgE reactivity to the sera described in example 1 were used to assess the IgE reactivity of each of these preparations. According to table 9, all preparations had similar IgE reactivity to these sera which was also in accordance with the IgE reactivity of the purified fraction containing native protein (table 6, fraction 2).
[0127] Further analysis of IgE reactivity using sera from 35 horse dander sensitized subjects was performed comparing recombinant Equ c s ab with the native purified protein fraction 2 from RPC (
[0128] There was a good agreement (r=0.99) between IgE binding to purified native Equ c s and recombinant Equ c s (
Example 8: Assessment of IgE Binding Activity of nEqu c 1, nEqu c 2, nEqu c 3, nEqu c 4, Equ c 15k and Equ c s in a Cohort of Horse Allergic Patients
[0129] Sera from 25 horse allergic subjects from Spain (n=20) and Sweden (n=5) were used in the study. All patients had a doctors' diagnosis of horse allergy with symptoms such as asthma, rhinoconjunctivitis and urticaria, and a positive skin prick test to horse dander extract. All samples and clinical data were collected under the approval of the local ethics committee at each center contributing to the biobank in which the samples and data had been deposited.
[0130] The levels of specific IgE antibodies to horse dander extract, nEqu c 1, nEqu c 2, nEqu c 3 and nEqu c 4, rEqu c 15k and rEqu c s ab among the 25 horse allergic subjects were determined using ImmunoCAP™ (
[0131] Of the 25 sera tested, 13 (52%) showed an IgE response ≧0.35 kU.sub.A/L to rEqu c s whereas 12 (48%) had IgE reactivity to rEqu c 15k, 16 (64%) to nEqu c 2 and 19 (76%) to nEqu c 1. Both nEqu c 3 and nEqu c 4/5 appeared as minor allergens among the subjects studied, binding IgE ab from only 5 (20%) and 7 (28%) of the tested sera, respectively. In this study cohort none of the patients reacted exclusively to rEqu c s whereas four (16%) and two of the 25 sera reacted exclusively to Equ c 15k and Equ c 1, respectively. On average among all Equ c 15k-reactive sera, the concentration of IgE antibody to Equ c s amounted to 30% of that to horse dander.
[0132] The corresponding relative concentration of IgE antibody to nEqu c 1 was 52%, whereas for nEqu c 2, nEqu c 3, nEqu c 4/5 and Equ c 15k the relative concentrations were 35%, 69%, 9% and 37%, respectively, among sera specifically reactive to those allergens. Twenty-four of the 25 sera showed IgE antibody binding to horse dander extract. All of those sera showed binding to at least one of the five individual horse allergens tested. The sum of the IgE binding levels to the individual component matched or exceeded that to horse dander extract.
Example 9: Cross Reactivity Between Equ c s and Secretoglobin from Cat, the Major Cat Allergen Fel d 1
[0133] Since the unknown IgE reactivity that was the starting point of this study was inhibited by Fel d 1, the relationship between recombinant Equ c s and Fel d 1 was investigated. The levels of IgE binding to Fel d 1 was evaluated in sera of 35 horse dander sensitized subjects, including those 25 horse allergic patients described in Example 8. There was significant correlation (r=0.92) between the IgE levels to recombinant Equ c s and rFel d 1 (
[0134] In order to further investigate the relationship between Equ c s and Fel d 1, the five sera used in example 1 were tested for cross-inhibition, using both horse dander extract, rFel d 1 and rEqu c s on solid phase as well as rEqu c 15k, rEqu c s and rFel d 1 as inhibitors at a final concentration of 100 μg/mL. As an inhibition control buffer, 0.1 M sodium phosphate buffer, pH 7.4, containing 0.3% human serum albumin, was used. Means of duplicate determinations of each inhibition were calculated and the fraction of inhibition was calculated as the fraction of the binding using inhibition control buffer that could be quenched with each inhibitor. In these selected sera, inhibition of binding to horse dander extract could only be achieved by Fel d 1 and Equ c s indicating that Equ c s indeed is the unknown protein in horse dander extract that is accountable for the binding of these sera (Table 11A). Binding to Fel d 1 immunoCAP can be inhibited by Fel d 1 itself but not by Equ c s (Table 11B) whereas binding to Equ c s could be inhibited by both Fel d 1 and Equ c s (Table 11C). This demonstrates that the IgE binding between Fel d 1 and Equ c s is indeed cross reactive as both the high extent of sequence homology between the two proteins (
Example 10: Assessment of IgE-Binding Properties of a Variant or Fragment (Analyte) of an Allergenic Protein
[0135] The original allergenic protein, in this case Equ c s, is immobilized to a solid support. Serum samples from at least three representative human patients sensitized to the relevant species and showing IgE reactivity to the original allergenic protein from that species are incubated for 2 h at room temperature with the analyte 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 solid supports carrying immobilized Equ c s to study whether preincubation with the variant or fragment of Equ c s specifically inhibits or significantly lowers IgE binding.
TABLE-US-00011 TABLE 1 IgE binding characteristics of sera utilised for detection of an unknown horse dander component. rEqu c CDE rFel d 1 HDE rEqu c 1 nEqu c 2 nEqu c 3 nEqu c 4 15K Serum kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L A >100 >100 12.0 0.19 0.46 0.05 0.11 0.08 B 97.0 95.1 11.4 0.21 0.32 0.07 0.16 0.08 C 87.9 >100 10.9 0.13 0.21 0.03 0.08 0.06 D 61.9 69.0 7.36 0.11 0.16 0.04 0.09 0.06 E 67.6 61.0 6.28 0.02 0.04 0.14 0.02 0.00 CDE—cat dander extract HDE—horse dander extract
TABLE-US-00012 TABLE 2 Inhibition of IgE binding to a) cat dander extract and b) horse dander extract, using the inhibitors Equ c 15k, Fel d 1 and Fel d 7. Concentration Inhibition Serum Inhibitor (kUA/L) (%) a) binding to cat dander extract solid phase A buffer 73.3 0 Equ c 15k 74.6 −2 Fel d 1 13.5 82 Fel d 7 76.2 −4 B buffer 59.7 0 Equ c 15k 56.4 6 Fel d 1 8.5 86 Fel d 7 58.7 2 C buffer 55.9 0 Equ c 15k 57.2 −2 Fel d 1 9.06 84 Fel d 7 57.6 −3 D buffer 39.1 0 Equ c 15k 39.1 0 Fel d 1 6.17 84 Fel d 7 37.7 4 E buffer 53.6 0 Equ c 15k 51.2 4 Fel d 1 19.0 65 Fel d 7 56.7 −6 b) binding to horse dander extract solid phase A buffer 7.46 0 Equ c 15k 7.87 −5 Fel d 1 0.12 98 Fel d 7 7.99 −7 B buffer 6.42 0 Equ c 15k 6.57 −2 Fel d 1 0.22 97 Fel d 7 6.84 −6 C buffer 6.27 0 Equ c 15k 5.97 5 Fel d 1 0.11 98 Fel d 7 6.09 3 D buffer 4.20 0 Equ c 15k 4.04 4 Fel d 1 0.10 98 Fel d 7 4.42 −5 E buffer 4.64 0 Equ c 15k 4.56 2 Fel d 1 0.06 99 Fel d 7 4.70 −1
TABLE-US-00013 TABLE 3 IgE binding of detector sera to immobilised fractions from SEC chromatography of horse dander extract Fraction 18 22 26 30 34 38 Serum kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L A 18.9 16.4 18.3 10.1 4.01 1.78 B 12.4 11.5 11.8 7.02 3.04 1.53 C 15.2 12.3 12.2 7.25 3.02 1.46 D 10.7 8.79 8.44 5.40 2.22 1.02 E 11.0 9.31 9.24 6.45 2.49 0.77
TABLE-US-00014 TABLE 4 IgE binding of detector sera to immobilised fractions from HIC chromatography of an enriched fraction from horse dander extract Fraction 8 17 23 36 39 42 60 Sera kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L A 0.21 0.25 0.44 2.67 3.99 11.9 22.9 B 0.29 0.65 0.55 2.69 3.15 9.43 16.8 C 0.17 0.22 0.30 2.03 3.25 9.98 18.0 D 0.17 0.19 0.26 1.59 2.27 6.62 11.4 E 0.06 0.16 0.19 1.15 2.20 8.32 13.9
TABLE-US-00015 TABLE 5 IgE binding of detector sera to immobilised fractions from anion exchange chromatography of an enriched fraction from horse dander extract. a) Immobilised fractions at dilution 1:4 Fraction B3 B2 C1 C2 C5 C8 D9 Sera kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L kU.sub.A/L A 25.7 25.5 23.5 23.3 20.3 20.5 5.2 B 18.1 17.9 17.9 16.9 16.2 16.3 4.0 C 17.1 16.8 16.8 16.7 17.1 16.2 3.8 D 11.6 11.7 11.7 11.2 11.4 10.3 2.6 E 14.9 14.4 14.7 13.9 14.5 12.1 2.0 b) Immobilised pools at dilution 1:20 Fraction B4-B1 C1-C4 C5-C11 Sera kU.sub.A/L kU.sub.A/L kU.sub.A/L A 10.71 9.00 6.50 B 8.92 7.67 5.07 C 8.46 6.72 4.97 D 5.44 4.48 3.26 E 5.74 4.46 3.00
TABLE-US-00016 TABLE 6 IgE binding of detector sera to immobilised fractions from RPC chromatography of an enriched fraction from horse dander extract. Fraction 1 2 3 Sera kU.sub.A/L kU.sub.A/L kU.sub.A/L A 17.8 21.1 5.52 B 13.2 15.1 4.41 C 13.5 15.2 4.14 D 9.68 11.0 2.97 E 8.10 9.99 1.92
TABLE-US-00017 TABLE 7 Peptide fragments matching the theoretical masses of trypsin cleaved Equ c s from in gel digestion of a) 5 kDa b and b) 10 kDa band of reduced sample and c) 18 kDa band of non-reduced sample. m/z m/z Equ c s SEQ ID measured theoretical chain range peptide NO: a) 2609.29 2609.28 1 23-45 -.DICPAVKEDVNIFLTGTPDDYVK.K 17 1825.85 1825.88 1 30-45 K.EDVNIFLTGTPDDYVK.K 18 1953.97 1953.98 1 30-46 K.EDVNIFLTGTPDDYVKK.V 19 908.50 908.49 1 46-52 K.KVSQYQR.N 20 780.39 780.39 1 47-52 K.VSQYQR.N 21 1068.59 1068.6 1 53-62 R.NPVILANAEK.I 22 1659.84 1659.88 1 71-85 K.LTAEDKENALSVLEK.I 23 891.39 891.35 1 86-92 K.IYSSDFC.- 24 b) 1697.84 1697.89 2 24-39 -CPSFYAVLGVLSLGSK.T 25 1510.67 1510.72 2 62-74 K.IQDCYNEAGVITK.I 26 c) 2609.31 2609.28 1 23-45 -.DICPAVKEDVNIFLTGTPDDYVK.K 27 1825.86 1825.88 1 30-45 K.EDVNIFLTGTPDDYVK.K 28 1953.93 1953.98 1 30-46 K.EDVNIFLTGTPDDYVKK.V 29 908.46 908.49 1 46-52 K.KVSQYQR.N 30 1068.56 1068.6 1 53-62 R.NPVILANAEK.I 31 1659.84 1659.88 1 71-85 K.LTAEDKENALSVLEK.I 32 1510.69 1510.72 2 62-74 K.IQDCYNEAGVITK.I 33
TABLE-US-00018 TABLE 8 Peptides identified by MS/MS from in-solution digested fraction from RPC. SEQ m/z m/z MS/MS Equ c s ID measured theoretical score chain range peptide NO: 802.34 802.41 22.2 1 23-29 -.DICPAVK.E 34 1825.96 1825.88 88.0 1 30-45 K.EDVNIFLTGTPDDYVK.K 35 908.46 908.49 55.3 1 46-52 K.KVSQYQR.N 36 780.34 780.39 25.9 1 47-52 K.VSQYQR.N 37 1068.58 1068.6 75.9 1 53-62 R.NPVILANAEK.I 38 1659.89 1659.88 82.4 1 71-85 K.LTAEDKENALSVLEK.I 39 891.31 891.35 14.8 1 86-92 K.IYSSDFC.- 40 1510.75 1510.72 127.2 2 62-74 K.IQDCYNEAGVITK.I 41 1697.90 1697.89 128.3 2 24-39 -CPSFYAVLGVLSLGSK.T 42 2573.50 2573.32 53.4 2 75-98 K.ISDLIIMGTITTSPECISHALSTL.T* 43 *Peptide identified by semitrypsin cleavage of protein.
TABLE-US-00019 TABLE 9 IgE reactivity of rEqu s c preparations peak 1 peak 2 peak 1 peak 2 rEqu c s ba rEqu c s ba rEqu c s ab rEqu c s ab Sera kU.sub.A/l kU.sub.A/l kU.sub.A/l kU.sub.A/l A 24.87 23.09 23.40 23.12 B 15.70 15.02 15.61 15.23 C 15.15 15.34 16.77 15.57 D 15.41 15.59 16.10 15.50 E 13.16 12.99 13.05 13.23
TABLE-US-00020 TABLE 10 IgE reactivity of 25 horse allergic patients Patient no symptoms Country e3 nEqu c 1 nEqu c 2 nEqu c 3 nEqu c 4 rEqu c 15k rEqu c s 1 Rhin SE 1.55 0.06 0.21 0.12 0.30 1.31 0.14 2 Rhin, astm SE 1.28 1.24 0.56 0.00 0.16 0.03 0.48 3 Rhin ES 4.79 1.42 0.13 0.00 0.04 1.89 0.05 4 Rhin, astm ES 5.87 4.96 2.32 0.07 0.53 0.16 1.14 5 Rhin, astm ES 1.79 1.28 0.26 0.01 0.15 0.04 1.12 6 Rhin, astm ES 8.74 5.41 5.56 0.00 0.34 0.02 0.80 7 Rhin, astm ES 0.21 0.00 0.02 0.00 0.02 0.20 0.06 8 Rhin, astm ES 4.55 1.41 2.02 0.00 0.53 0.86 0.10 9 Rhin ES 0.55 0.00 0.01 0.00 0.02 0.63 0.06 10 astm, urt, a ES 17.31 6.20 2.67 11.90 3.11 6.07 0.08 11 Rhin ES 16.62 1.30 15.15 5.04 0.86 0.10 1.72 12 Rhin, urt ES 13.49 2.91 1.19 0.03 0.30 12.96 1.31 13 hin, astm, u SE 26.19 11.04 7.48 0.05 2.94 5.68 1.80 14 Rhin SE 6.58 3.42 1.08 0.01 0.48 1.16 2.23 15 Rhin, astm SE 7.01 0.03 0.04 0.04 0.20 7.45 0.13 16 Rhin ES 6.78 5.77 0.95 9.43 0.28 0.02 0.97 17 Rhin ES 28.73 21.92 5.89 33.75 1.19 0.24 9.25 18 Rhin, urt ES 13.81 5.44 14.10 0.05 0.13 0.07 0.14 19 Rhin, astm ES 5.18 0.06 0.08 0.06 0.18 5.81 1.54 20 Rhin, astm ES 0.78 0.76 0.09 0.01 0.02 0.02 0.06 21 Rhin, astm ES 1.96 1.63 0.58 0.01 0.16 0.04 0.32 22 Rhin, urt ES 1.28 0.24 1.75 0.01 0.02 0.06 0.23 23 Rhin, astm ES 6.94 2.49 0.30 0.00 0.10 1.36 4.02 24 Rhin, astm ES 3.18 1.46 1.31 0.00 0.13 0.60 0.05 25 Rhin ES 7.78 5.46 1.32 4.61 0.08 0.03 4.99
TABLE-US-00021 TABLE 11 Inhibition of IgE binding to a) horse dander extract, b) rFel d 1 and c) rEqu c s solid phase, using the inhibitors Equ c 15k, Equ c s and Fel d 1. Concentration Inhibition Serum Inhibitor (kUA/L) (%) a) binding to horse dander extract solid phase A buffer 6.71 0 Equ c 15k 7.40 −10 Equ c s 0.27 96 Fel d 1 0.13 98 B buffer 5.03 0 Equ c 15k 5.10 −2 Equ c s 0.33 93 Fel d 1 0.20 96 C buffer 5.49 0 Equ c 15k 5.50 0 Equ c s 0.25 96 Fel d 1 0.12 98 D buffer 5.01 0 Equ c 15k 5.32 −6 Equ c s 0.22 96 Fel d 1 0.13 97 E buffer 3.70 0 Equ c 15k 4.06 −10 Equ c s 0.09 98 Fel d 1 0.06 98 b) binding to rFel d 1 solid phase A buffer 54.97 0 Equ c 15k 57.45 −5 Equ c s 58.70 −7 Fel d 1 5.22 91 B buffer 42.07 0 Equ c 15k 43.80 −4 Equ c s 43.20 −3 Fel d 1 3.87 91 C buffer 47.30 0 Equ c 15k 49.98 −6 Equ c s 48.35 −2 Fel d 1 4.01 92 D buffer 44.15 0 Equ c 15k 45.25 −2 Equ c s 41.90 5 Fel d 1 3.85 91 E buffer 32.38 0 Equ c 15k 33.32 −3 Equ c s 29.99 7 Fel d 1 2.02 94 c) binding to rEqu c s solid phase A buffer 13.39 0 Equ c 15k 11.73 12 Equ c s 5.30 60 Fel d 1 0.59 96 B buffer 9.08 0 Equ c 15k 8.63 5 Equ c s 3.47 62 Fel d 1 0.45 95 C buffer 10.05 0 Equ c 15k 9.37 7 Equ c s 3.82 62 Fel d 1 0.40 96 D buffer 9.22 0 Equ c 15k 8.44 8 Equ c s 3.76 59 Fel d 1 0.40 96 E buffer 6.60 0 Equ c 15k 6.33 4 Equ c s 1.59 76 Fel d 1 0.24 96
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