Abstract:
The present invention relates to the use of an isolated allergen component in a serological assay for improved precision in the identification of the actual sensitizer in diagnosis of allergy. This enables not only adequate measures for avoidance of the causative agent but also appropriate selection of treatment of the allergic disease.

Description:
FIELD OF THE INVENTION  
         [0001]    The present invention relates to the use of an isolated allergen component in a serological assay for improved precision in identification sensitizing allergen source in the diagnosis of allergy. This enables not only adequate measures for avoidance of the causative agent but also appropriate selection of treatment of the allergic disease.  
         BACKGROUND OF THE INVENTION  
         [0002]    About 20% of the western world population suffer from Type I allergy, a hypersensitivity disease involving the formation of IgE antibodies against otherwise harmless molecular structures present in the environment, i.e. allergens. The symptoms of Type I allergy (allergic rhinitis, conjunctivitis and allergic asthma) are mainly caused by the crosslinking of effector-cell bound specific IgE antibodies by allergen molecules, which triggers the release of biological mediators such as histamine and leukotriens that give rise to the tissue reaction.  
           [0003]    Diagnosis of allergy includes measures to identify the source of sensitizing substances. For this purpose, common practise is to use extracts of allergen sources either for in vivo challenge, typically in the skin and scoring for tissue reaction, or for serological testing by measuring specific IgE antibodies. In the latter modality, serum from a blood sample is used for quantitative and objective testing against any number of suspected allergens without subjecting the patient to offending allergens. The identifying precision of diagnostic methods utilizing crude allergen extracts is, however, hampered by the extensive patterns of serological relationships that exist between molecular structures present in closely and distantly related species producing allergens.  
           [0004]    Thus, sensitization to a particular allergen source can lead to scropositivity to a range of other allergenic materials which the patient may never have been in contact with. Such serological cross-reactivity may or may not be clinically relevant (i.e. capable of eliciting allergic symptom) and can act to confound the identification of offending allergen which forms the basis for meaningful advice on allergen avoidance and, in particular, for the choice of allergen extract for immunotherapy.  
           [0005]    Weed pollens constitute important allergen sources worldwide, including prominent examples such as ragweed (Ambrosia spp.), mugwort (Artemisia vulgaris) and wall pellitory (Parietaria spp). Some but not all of the allergens present in pollen of any particular weed species are represented by structurally similar homologous in other species and therefore show some degree of serological cross-reactivity. Thus, depending on the composition of antibody specificities generated by the immunological response in an allergic individual, sensitization to one weed species may lead to serological test positivity also to other species. Serological testing will in such cases of apparent multisensitization generate results that are ambiguous in terms of identification of actual sensitizer.  
           [0006]    Using total allergen extracts it is therefore often difficult or impossible to unambiguously determine the allergen source responsible for sensitization and to ensure an optimal choice of selective immunotherapy. Selection of an appropriate allergen source for immunotherapy, which accurately reflects the primary or actual sensitizer, is highly desirable with respect to treatment efficacy, risk of acute side effects and induced additional sensitization, as well as to cost saving and inconvenience to the patient undergoing treatment.  
           [0007]    EP 0707 065 describes recombinant Parietaria proteins and derived peptides identified as being allergenic. In EP 0707 065, these peptides are contemplated for use in both therapy and diagnosis of Parietaria pollen-induced allergy. However, the utility of these proteins/peptides as reagents to distinguish between genuine Parietaria pollen sensitization and cross-reaction-mediated seropositivity to Parietaria pollen extract is not demonstrated or discussed.  
         SUMMARY OF THE INVENTION  
         [0008]    According to the present invention stringent differentiation between different weed pollen allergies is possible for the first time, significantly improving the criteria for accurate choice of selective immunotherapy.  
           [0009]    The present invention provides a new use of an isolated allergen component of limited cross-reactivity, to serologically determine sensitizing allergen source, among a variety of possible allergen sources, which higher level of accuracy than is possible using crude allergen extracts. This enables enhanced precision in the choice of allergen extract for selective treatment of a disorder involving an allergen, such as Type I allergy. The treatment may comprise immunotherapy of pharmacotherapy.  
           [0010]    Thus, the invention provides use of a pure allergen component of limited or no cross-reactivity to serologically identify with improved accuracy the actual sensitizing allergen source among a variety of possible allergen sources containing cross-reactive proteins or epitopes. The use of the invention is, for example, valuable for selection of treatment of a disorder involving extract, proteins or peptides derived from said actual allergenic sensitizer.  
           [0011]    The allergen component is derived from pollen of a plant species, preferably a weed species, such as mugwort, ragweed or wall pellitory.  
           [0012]    A preferred species is  Parietaria judaica , especially wherein the allergen component is Par j 1 or Par j 2 (1, 2). The method used to generate these components is not crucial for the invention and may be synthetic, recombinant or native. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0013]    The present invention will now be described in association with an exemplifying allergen component, i.e. Par j 2.  
         [0014]    The present inventors investigated whether recombinant Par j 2, a major  Parietaria judaica  pollen allergen, can be used as a diagnostic tool to identify allergic patients with primary sensitization to Parietaria, a mediterranean weed. The present inventors analysed the serum IgE reactivity of patients reactive to total parietaria extracts from Austria (n=42), Scandinavia (n=8), USA (n=18) and Italy (n=37) to ragweed, mugwort, Parietaria pollen extracts and rPar j 2 by immunoblot as well as by CAP RAST measurements. It was found that almost all patients from Scandinavia, USA and Austria contained IgE antibodies to ragweed, mugwort and Parietaria pollen extracts. No serum from Scandinavia and USA and only four Austrian scra, but 81% of the mediterranean patients reacted with rPar j 2. rPar j 2 can therefore be used as a marker allergen to identify allergic patients with a mediterranean sensitization profile who were primarily sensitized to Parietaria pollen. rPar j 2 may thus be a useful diagnostic tool for the selection of suitable weed pollen allergen extracts for specific immunotherapy of weed pollen allergic patients.  
       MATERIALS AND METHODS  
     Patients Sera  
       [0015]    Weed pollen allergic patients from Austria (n=42), Scandinavia (n=8), USA (n=18) and Mediterranian (n=37) were characterized by skin reactivity and immunoblot. The presence of serum IgE Abs specific for ragweed, mugwort, Parietaria pollen extracts and rPar j 2 was confirmed by RAST (radioallergosorbent test) analysis (Pharmacia, Uppsala, Sweden).  
       Allergen Extracts, Western Blotting  
       [0016]    Pollen from mugwort ( Artemisia vulgaris ) and Parietaria ( Parietaria judaica ) were obtained from Allergon AB (Välinge, Sweden) and stored at 4° C. until use. Two grams of pollen was extracted in 50 ml of distilled water for 2 hours at room temperature. The protein extracts were then centrifuged at 30,000 g for 30 minutes at 4° C. Supernatants were frozen, lyophilized, and checked for protein quantity and quality of proteins by SDS-PAGE and Coomassie blue staining (Bradford et al. 1976). Comparable amounts of each extract (220 μg/cm) were separated by 12% SDS-PAGE (Fling et al. 1986) and blotted onto nitrocellulose (Schleicher &amp; Schuell, Dassel, Germany) (Towbin et al. 1979).  
       rPar j 2 Production  
       [0017]    The Par j 2 protein was expressed in  E. coli  strain BL21 from a cDNA described by Duro et al. (2), cloned in the pET-23a expression vector. Culture medium was inoculated 1 to 500 with an overnight culture and first grown at 30° C. to mid-log phase. The incubation temperature was then raised to 42° C. for 45 min, followed by 4 hrs at 30° C. before harvest. Cells were collected by centrifugation at 10 000 g for 10 min at +4° C. and resuspended in 5 ml of buffer A (20 mM Tris-HCl p11 8.0, 0.5 M NaCl, 5 mM imidazole) per gram (fresh weight) of cells. The resuspended cells were ruptured by sonication while kept on ice, followed by centrifugation to remove solid material. The supernatant was loaded onto a Ni 2+ -charged 5 ml HiTrap Chelating column (Amersham Pharmacia Biotech, Uppsala, Sweden) for IMAC. The column was washed with 10 volumes of buffer A containing 20 mM imidazole and elution was performed with a 20-500 mM gradient of imidazole in buffer A Fractions containing pure rPar j 2 protein were pooled and subjected to buffer exchange to 0.15 M NaCl using a Sephadex G-25 column (Amersham Pharmacia Biotech, Uppsala, Sweden). The concentration of rPar j 2 was determined from absorbance at 280 nm, using a calculated absorption coefficient of 0.24 per mg/ml. The protein was divided into aliquots and stored at −20° C. until use.  
       Analysis of IgE-Binding by rPar j 2 Using Pharmacia CAP System  
       [0018]    In vitro IgE antibody binding by rPar j 2 was examined in Pharmacia CAP System, a clinically used allergy diagnostic immunoassay system Experimental ImmunoCAP tests were prepared by covalent immobilisation of the purified allergen onto activated cellulose at a concentration chosen so as to achieve an adequate linear measuring range and a background for negative sera below the conventional cut-off value. Serum testing was performed according to the recommendations of the immunoassay system manufacturer (Pharmacia Diagnostics AB, Uppsala, Sweden). Statistical parameters attesting the quality of the assays were calculated using system software  
       Immunoblotting  
       [0019]    Twenty weed pollen allergic patients from Austria, thirty-two from Italy and (for control purposes) one nonatopic individual, were diluted 1:10 in buffer A (50 mM Na phosphate, pH 7.5, 0.5% w/v bovine serum albumin (BSA), 0.5% v/v Tween 20, 0.05% NaN 3 ).  
         [0020]    Sera were exposed to nitrocellulose-blotted mugwort and Parietaria pollen extracts. Bound IgE was detected with  125 J-labeled anti-human IgE antibodies (Valenta et al. 1992) and visualized by autoradiography using KODAK X-OMAT films and intensifying screens (Kodak, Heidelberg, Germany).  
       IgE Inhibition Experiments  
       [0021]    The presence of cross-reactive allergens/epitopes in mugwort and Parietaria extracts were investigated by IgE immunoblot inhibition experiments. Six mugwort allergic patients from the Austrian population were diluted {fraction (1/10)} and preadsorbed with 100 μg/ml mugwort extract, 100 μg/ml Parietaria extract, 10 μg/ml rPhl p 7, 10 μg/ml Bet v 2 and for control purposes with 10 μg/ml BSA overnight at 4° C. Preadsorbed sera were then exposed to nitrocellulose-blotted mugwort and Parietaria extract. Bound IgE Abs were detected with  125 J-labeled anti-human IgE antibodies (Pharmacia, Uppsala, Sweden).  
       Skin Prick Testing  
       [0022]    After informed consent was obtained, skin-prick tests were performed on the forearms of ten weed pollen allergic patients, and for control purposes of a nonallergic individual. Drops of ragweed pollen extract, mugwort pollen extract, Parietaria pollen extract, histamine (1 μg/ml), and sodium chloride solutions (Soluprick; ALK, Horsholm, Sweden) were pricked with sterile lancets (ALK) as described (Vrtala et al. 1997). The skin reactions were recorded 20 min after testing by photography and by transferring the ball point pen-surrounded wheal area with scotch tape to paper. The mean wheal diameters displayed in Table 5 were determined as follows: Dm=(D1+D2)/2. D1 and D2 represent the maximal longitudinal and transversal diameter of the wheal reaction in mm, respectively.  
         [0023]    [0023]FIG. 1: Different IgE reactivity profiles of Austrian and Mediterranean sera to blotted Parietaria extract.  
         [0024]    Serum IgE reactivity of weed pollen allergic patients from Austria (A, B; n=21) and Italy (C, D; n=33) was tested to nitrocellulose-blotted mugwort (A, C) and parietaria (B, D) pollen extracts. Sixteen out of 20 Austrian weed pollen allergic patients showed IgE reactivity to proteins of different molecular weights to blotted Parietaria pollen extract (B).  
         [0025]    In contrast, the mediterranean population shows a different reactivity-profile. Most patients reacted to Par j 2 at 10 kDa.  
         [0026]    [0026]FIG. 2: IgE inhibition experiment.  
         [0027]    Six sera (A, B, C, D, E, F) from Austrian weed pollen allergic patients were preincubated either with mugwort pollen extract, Parietaria pollen extract, rPhl p 7, rBet v 2 or BSA and then exposed to nitrocellulose-blotted mugwort and Parietaria pollen extract. Mugwort and parietaria pollen extracts contain cross-reactive allergens of high molecular weight (25-100 kDa), profilin and two EF-hand allergens.  
         [0028]    [0028]FIG. 3: Quantitative analysis of serum IgE antibody binding to crude extracts of Parietaria pollen and the allergen component rPar j 2.  
         [0029]    Graphs are based on IgE antibody concentrations detailed in Tables 1-3.  
         [0030]    Serum IgE to Par j 2 was measured for three groups of individuals, A=Austrian, Scandinavian and American, B=Mediterranean, and C=All these together.  
         [0031]    The results show predominant sensitization to rPar j 2 in the patient population that originates from the region where Parietaria occurs, and virtually no sensitization to this protein in the populations that live outside the natural distribution area of Parietaria.  
         [0032]    Tables 1-4. Detection of IgE antibodies to ragweed, mugwort, Parietaria and rPar j 2 by CAP RAST measurements in sera from different weed pollen allergic populations.  
         [0033]    CAP RAST measurements show that almost all weed pollen allergic patients from Scandinavia, USA and Austria contain IgE antibodies to Parietaria pollen extract. No serum from Scandinavia and USA and only four (9.5%) Austrian sera, reacted with rPar j 2. By constrast, 81% of the mediterranean sera contained IgE anti-rPar j 2.  8   
                                     TABLE 1                           Austria       specific IgE:kUA/l            patient   ragweed   mugwort   Parietaria   rPar j 2                1   neg   2.41   neg   neg        2   10      4.18   5     neg        3   0.9    5.31   0.80   neg        4   4.72   3.26   0.66   neg        5   0.68   2.7    0.52   neg        6   17.8    4.86   0.78   neg        7   53.4    46.7    6.99   neg        8   20.2    14.9    8.73   neg        9   1.15   2.23   0.60   neg       10   0.97   6.45   neg   neg       11   1.06   2.4    2.28   1.25       12   14.9    7.68   4.35   neg       13   2.46   neg   neg   neg       14   3.62   2.6    0.92   neg       15   23      7.92   1.33   neg       16   1.99   12.3    neg   neg       17   4.83   2.27   0.74   neg       18   neg   2.03   neg   neg       19   5.49   3.24   3.13   neg       20   0.83   2.21   neg   neg       21   2.27   2.32   0.94   neg       22   4.34   4.75   2.37   neg       23   5.29   5.71   11.9    neg       24   3.52   12.8    neg   neg       25   2.17   0.8    1.26   neg       26   3.92   3.56   1.84   neg       27   3.23   2.39   4.74   neg       28   21.6    19.4    15.9    0.49       29   2.95   10.6    neg   neg       30   5.1    3.75   1.84   neg       31   4.14   3.06   1.85   neg       32   neg   3.02   neg   neg       33   5.24   3.33   3.70   neg       34   &gt;100       15.2    11.8    0.56       35   17.3    37.3   neg   neg       36   7.14   4.03   4.81   neg       37   1.19   18.5    neg   neg       38   36      9.21   25      1.80       39   6.23   6.51   1.44   neg       40   0.39   5.95   neg   neg       41   6.94   5.3    2.14   neg       42   11.2    6.17   4.22   neg       average   10.72    7.79   4.42   1.03                  
 
         [0034]    [0034]                                                                                             TABLE 2                           specific IgE:kUA/l            patient   ragweed   mugwort   parietaire   rPar j 2                    Scandinavia            1   14.82    21.22    2     neg       2   6.66   24.74    1.94   neg       3   2.75   4.81   6.48   neg       4   3.30   2.29   2.77   neg       5   10.95    5.54   7.50   neg       6   5.15   3.63   3.53   neg       7   9.70   6.43   8.76   neg       8   5.48   3.70   2.36   neg       average   7.35   9.05   4.42   —            USA            1   neg   0.45   2.35   neg       2   13.49    7.47   4.21   neg       3   3.06   1.72   1.93   neg       4   0.39   neg   3.47   neg       5   9.09   3.89   7.62   neg       6   3.03   1.86   5.12   neg       7   1.95   1.37   3.22   neg       8   111.11    5.10   4.31   neg       9   8.37   12.72    3.39   neg       10    22.16    11.01    3.19   neg       11    8.03   3.91   4.62   neg       12    3.45   4.35   3.14   neg       13    8.39   6.63   8.05   neg       14    14.16    5.99   10.29    neg       15    21.52    5.64   6.37   neg       16    42.60    9.71   4.97   neg       17    5.00   4.84   3.37   neg       18    90.17    6.80   6.92   neg       average   21.53    5.5    4.81   —                    
         [0035]    [0035]                                     TABLE 3                           Mediterranean       specific IgE:kUA/l            patient   ragweed   mugwort   parietaire   rPar j 2                1   4.44    3.01    3.73   neg        2   neg   neg   16.72   10.97        3   1.55   neg   113.79    64.19        4   neg    0.53   80.52   48.67        5   0.67   neg    4.60    2.34        6   2.92    0.37   37.33   14.48        7   neg   neg   17.36   14.89        8   neg   neg    3.29    3.53        9   0.74    1.07    5.65    5.71       10   neg   neg   53.65   51.61       11   neg    0.41   19.22   neg       12   neg   neg   84.12   45.52       13   neg    0.51    6.37    2.97       14   neg   neg   25.22   16.27       15   neg   neg   12.93    8.28       16   5.61    2.89    5.35   neg       17   neg   neg   21.10   15.04       18   neg   neg   11.08   10.26       19   neg   neg    8.34    5.60       20   neg   neg   11.33    7.84       21   neg   neg   33.89   20.92       22   1.92   neg   42.24   25.89       23   2.44    1.21   34.20   28.76       24   neg   neg   10.14    6.63       25   neg   neg   36.77   21.40       26   1.91   neg   30.90   19.46       27   neg   neg    7.90    5.53       28   0.95   neg   34.41   23.71       29   neg   neg   44.36   34.96       30   neg   neg    5.92    4.07       31   neg   neg    5.00    2.66       32   neg   neg   34.93   26.08       33   2.11   neg   122.59    78.49       34   33.42    11.67   46.94   neg       35   22.38     8.19   15.92   neg       36   45.24    23.90   40.74   neg       37   14.17    11.67    9.74   neg       average   9.36    5.45   29.95   20.89                    
         [0036]    [0036]                                           TABLE 4                               % of parietaire   % of rPar j 2       average   IgE for           origin   n   positive sera   reactive sera   ragweed   mugwort   parietaire   rPar j 2                   Austria   42    71     9.5   10.72   7.79   4.42    1.03       Scandinavia    8   100   —    7.35   9.05   4.42   —       USA   18   100   —   21.53   5.5    4.81   —       Mediterranian   37   100   81    9.36   5.45   29.95    20.89                    
         [0037]    [0037]                                                       TABLE 5                           Skin prick   testing           specific   IgE:kUA/l               2EF-hand       Individual   Ragweed   Mugwort   Parietaria   Histamine   Ragweed   Mugwort   Parietaria   rPar j 2   profilin   allergen                   1     6.5   7.5   4   5     3.52   12.8    —   —   +   n.d.       2   5   6     —   8.5   5.1    3.75   1.84   —   −   n.d.       3   11    9.5   —   8     &gt;100       15.2    11.8    0.56   −   n.d.       4     8.5   9       11.5    6.5   7.14   4.03   4.81   —   +   +       5   6   19.5    5   7.5   0.39   5.95   —   —   −   n.d.       6     5.5   8       3.5   6.5   6.94   5.3    2.14   —   +   n.d.       7     6.5   9.5   5   4     n.d.           +   n.d.       8   6   9.5     8.5   5.5   20.2    14.9    8.73   —   −   +       9   8   14       11.5    6.5   5.49   3.24   3.13   —   +   +       10    3   5     4   6     n.d.           +   n.d.       11    —   —   —   7     —   —   —   —   −   −                    
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