Quantitation of the major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products

Quantitation of the major fungal allergens, Alt a 1 and Asp f 1, in commercial allergenic products Lisa Vailes, MS,a Susheela Sridhara, PhD,a* Oliver Cromwell, PhD,b Bernhard Weber, PhD,b Michael Breitenbach, PhD,c and Martin Chapman, PhDa Charlottesville, Va, Reinbek, Germany, and Salzburg, Austria

Background: Alternaria is one of the most important fungi associated with allergic disease, whereas Aspergillus fumigatus is involved in a broad spectrum of pulmonary diseases. Currently, fungal extracts used for diagnosis in the United States are unstandardized, and their allergenic content cannot be compared directly. Objective: The goal of this study was to compare the variability of major allergen levels among US allergenic products derived from fungi: specifically, Alt a 1 levels in Alternaria alternata extracts, and Asp f 1 levels in A fumigatus extracts. Methods: A novel 2-site monoclonal antibody ELISA was used for measuring Alt a 1 using recombinant Alt a 1 as a standard. Asp f 1 was also measured by ELISA. Allergenic products produced by 8 US manufacturers over a 2-year period were compared, as were multiple lots produced by a single company. Results: Alt a 1 levels in Alternaria extracts from 8 companies produced in 1998 and 1999 ranged from less than 0.01 to 6.09 µg/mL (mean 1.4 ± 1.6 µg/mL, n = 15). In general, Alt a 1 levels were consistent within and between companies (1.4 ± 1.1 µg/mL, n = 27), with 21 of 32 (66%) of all extracts tested containing 0.7 to 2 µg/mL Alt a 1. Aspergillus extracts showed much greater variability in Asp f 1 levels, with extracts from 8 companies containing from less than 0.1 to 64 µg/mL Asp f 1 (mean 16.3 ± 23.9 µg/mL, n = 15). Overall variability was greater for Aspergillus products within and between manufacturers (22 ± 22 µg/mL Asp f 1, n = 20). Conclusions: ELISA-based assays for specific allergens showed greater consistency among allergenic products derived from Alternaria than from Aspergillus. These assays should facilitate improved quality control and standardization of fungal allergen extracts and lead to the development of more consistent products for clinical use. (J Allergy Clin Immunol 2001;107:641-6.)

From athe Asthma & Allergic Diseases Center, Department of Medicine, University of Virginia, Charlottesville, bAllergopharma, Joachim Ganzer KG, Reinbek; and cUniversity of Salzburg, Austria. Supported by NIH grants AI 34607 and AI 32557. Dr Sridhara was the recipient of a DBT Overseas Associateship from the Department of Biotechnology, New Delhi, India. *Dr Sridhara is currently at the Centre for Biochemical Technology, Delhi, India. Received for publication June 30, 2000; revised December 21, 2000; accepted for publication January 8, 2001. Reprint requests: Asthma & Allergic Diseases Center, Box 801355, Charlottesville, VA 22908-1355. Copyright © 2001 by Mosby, Inc. 0091-6749/2001 $35.00 + 0 1/81/114118 doi:10.1067/mai.2001.114118

Key words: Alternaria, Aspergillus, allergen exposure, recombinant allergens, fungi, asthma

Alternaria alternata is considered to be one of the most important fungi causing allergic disease in the United States, and sensitization to Alternaria allergens is associated with asthma.1-8 Alternaria sensitization was an independent risk factor associated with the development of wheezing and asthma in children and young adults (odds ratios 5-6.8), and skin test sensitivity to Alternaria was significantly associated with increased bronchial responsiveness.4,6-8 Alternaria was the major asthma-associated allergen in desert regions of the United States and Australia and has been reported to cause serious respiratory arrest and death in the US Midwest.9-12 Aspergillus species are important opportunistic fungi involved in a wide range of human respiratory diseases, including allergic asthma, rhinitis, sinusitis, aspergilloma, allergic bronchopulmonary aspergillosis, and systemic invasive aspergillosis of immunocompromised individuals.13,14 Aspergillus fumigatus in particular is responsible for 80% of Aspergillus infections in human beings. Most fungal allergen extracts used for diagnosis and immunotherapy contain large amounts of nonallergenic proteins. Variability of allergen extracts is a particular problem with molds because of interstrain differences, culture methods, the origin of the source material, and differences in extraction procedures.13,15-21 Attempts to assess allergen levels in Alternaria extracts have used techniques such as immunoelectrophoresis, RAST inhibition, and RIAs. These procedures are labor intensive and use polyclonal antibodies and human serum IgE, which are in short supply. 16,22,23 Currently, fungal allergen extracts manufactured in the United States are not standardized, and the allergen content of the extracts produced by different manufacturers cannot be directly compared. Several A alternata and A fumigatus allergens have been cloned. Alt a 1 and Asp f 1 are major allergens of A alternata and A fumigatus, respectively, and more than 80% of patients sensitized to the fungi have IgE antibody to these allergens.13,14,24-28 Alt a 1 is a heat-stable dimer of 28 kd, which dissociates into 14.5-kd and 16-kd subunits under reducing conditions.26,29,30 Asp f 1 is an 18kd protein showing homology to a family of cytotoxins and is thought to be a virulence factor for A fumigatus.14 641

642 Vailes et al

Abbreviation used rAlt a 1: Recombinant Alt a 1

Recently, Aden et al31 described the development of a novel 2-site mAb ELISA for the measurement of Alt a 1. The assay is unique in that a single mAb, which binds to a common epitope on both Alt a 1 monomers, is used as both capture and biotinylated secondary antibody. In the present study, 2-site ELISAs for Alt a 1 and Asp f 1 were used to compare Alt a 1 and Asp f 1 levels in commercially available, unstandardized A alternata and A fumigatus extracts, in reference extracts, and in fungal spores.

METHODS Allergen extracts Extracts of A alternata and A fumigatus were obtained from 8 manufacturers: Allergy Labs of Ohio, Allergy Labs of Oklahoma, Allermed, Bayer (now Hollister-Stier Laboratories), Center, Greer Laboratories, Meridian Biomedical Inc (ALK/Abelló), and Nelco. All extracts, purchased in 1998 and again in 1999, were prepared in 50% glycerine and labeled as 1:10 or 1:20 wt/vol. Full details of lot numbers and expiry dates are available on request. Six freeze-dried Alternaria extracts prepared as part of an allergen standardization study in 1983 were obtained from Dr John Yunginger (Mayo Clinic, Rochester, Minn).23 Bayer kindly provided 12 A fumigatus and 10 A alternata extracts for comparison.

ELISA for Alt a 1 mAb 121 (clone 121G 5G8) and Alternaria extract No. 43771 containing 8.5 µg natural Alt a 1 per vial (Allergopharma, Germany) were used to establish the ELISA.31 In brief, Immulon II plates were coated with 1 µg/mL mAb 121 in 0.05 mol/L carbonate bicarbonate buffer (pH 9.6). mAb 121 was biotinylated as previously described and used for detection of bound allergen.32 Recombinant Alt a 1 (rAlt a 1, lot No. 1 28.09.96) was obtained from Dr Michael Breitenbach (Vienna, Austria) and compared with natural Alt a 1 for use as a standard. Control curves were routinely carried out with rAlt a 1 at 0.2 to 100 ng/mL.

ELISA for Asp f 1 Asp f 1 was measured as described previously with anti-Asp f 1 mAb 4A6 for antigen capture and polyclonal rabbit anti-Asp f 1 for detection.14 Purified natural Asp f 1 (0.45-250 ng/mL) was used to form a standard curve.

Spore extracts Dried A alternata spores harvested in 1979 from Orange County, Calif, and stored in the dark at 4°C were kindly provided by Dr Don Hoffman. An extract was made by incubating 28 mg of spores in 0.5 mL PBS (pH 7.4) on a rotator for 30 minutes at room temperature. Spores in solution, washed from A alternata growing on agar plates, were kindly provided by Dr Linda Stetzenbach (University of Nevada, Las Vegas).

SDS-PAGE analysis Natural Alt a 1, rAlt a 1, and affinity-purified natural Asp f 1 were analyzed by silver stain on a high-density SDS-PAGE gel with the Pharmacia Phast Gel System.

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RESULTS Allergen analysis SDS-PAGE analysis showed that natural Alt a 1 exists as a 29-kd dimer but breaks down into 15-kd and 16-kd subunits under reducing conditions. rAlt a 1 migrates as an approximately 15-kd protein under reducing conditions but tends to dimerize during storage and shows both 29-kd and 15-kd bands under nonreducing conditions. Purified Asp f 1 migrates as a single band at 18 kd (Fig 1).

mAb ELISA for Alt a 1 Almost identical, parallel dose-response curves were obtained with either natural Alt a 1 or rAlt a 1 in the ELISA. Alternaria extracts from 4 different manufacturers also gave parallel dilution curves (Fig 2). Natural and recombinant allergen showed greater than 95% inhibition in both ELISA and RIA (data not shown). Because the binding of mAb 121 was the same for both natural and rAlt a 1, the ELISA was quantitated with rAlt a 1 as a standard, in control curves ranging from 0.2 to 100 ng/mL. The assay was highly specific for Alt a 1, and a panel of other fungal extracts gave negative results: Cladosporium, Phoma, Candida, Trichophyton, and Rhizopus (Greer Laboratories); Aspergillus species, Fusarium, Penicillium, Rhodofarula, and Stachybotrys (Dr Kurup, Milwaukee, Wis); and Curvularia and Epicoccum (Centre for Biochemical Technology, Delhi, India).

Comparison of Alt a 1 and Asp f 1 levels in allergenic products Alt a 1 concentrations were measured in 8 commercial A alternata extracts, which were purchased in 1998 and 1999. As a comparison, A fumigatus extracts from the same companies were assayed for Asp f 1 content. Alt a 1 was detected in all but 1 extract, with relatively consistent levels between extracts obtained in 1998 and 1999 for each company. Alt a 1 levels ranged from 0.01 to 6.09 µg/mL, with an average of 1.4 ± 1.6 µg/mL (n = 15) (Table I). Results were quite different for Aspergillus. Four companies produced products that had low or undetectable levels of Asp f 1 (<0.06 µg/mL), whereas others produced products that contained very high levels, ranging from 4 to 64 µg/mL (average 16.3 ± 23.9 µg/mL, n = 15). Intra-assay and interassay coefficients of variation were 5% and 33%, respectively, for the Alt a 1 ELISA and 8% and 17%, respectively, for the Asp f 1 ELISA. In the 1980s, an international collaborative study was carried out under the auspices of the WHO/IUIS Allergen Standardization Committee to develop an international reference standard of Alternaria.23 The Alternaria extracts used in that study, together with the proposed international reference, were assayed for Alt a 1 to provide a comparison of allergen levels with extracts currently in use. Alternaria extracts prepared and stored in 1983 had remarkably similar Alt a 1 levels (~1 µg/mL) to those manufactured in 1998 and 1999, with only 1 extract having undetectable allergen (Table II). These results suggest that at least with respect to Alt a 1 con-

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FIG 1. SDS-PAGE analysis of Alternaria and Aspergillus allergens. Lane 1, molecular weight markers; lane 2, natural Alt a 1, nonreduced; lane 3, natural Alt a 1, reduced; lane 4, rAlt a 1, nonreduced; lane 5, rAlt a 1, reduced; lane 6, natural Asp f 1, nonreduced.

FIG 2. Dose-response curves of natural and rAlt a 1 and commercial Alternaria extracts in the ELISA. , Natural Alt a 1; , rAlt a 1; ▲, Bayer; , Allergy Labs of Ohio; ■, Allermed; , Greer.

TABLE I. Allergen levels in commercial Alternaria and Aspergillus extracts Alt a 1 (µg/mL) in Alternaria extract

Asp f 1 (µg/mL) in Aspergillus extract

Manufacturer

1998

1999

1998

1999

Meridian (ALK/Abelló) Nelco Bayer Center

0.03 1.93 6.09 0.50

0.01 0.57 3.72 0.38

64.0 28.2 17.5 10.3

4.26 54.30 61.00 5.14

1.00 1.57 2.23 <0.01

0.73 1.35 0.95* 0.04

Greer Allermed Allergy Labs, Ohio Allergy Labs, Okla

0.06 0.05 0.04 0.04

0.04 0.01 0.04* NA

Bold facing indicates 4 companies having very high ASP f 1 levels as compared with the remaining 4 companies. NA, Not available. *Greer extract supplied.

tent, there has been little change in the production of Alternaria extracts during the past 17 years (although the results do not rule out the possibility that there may have been some degradation of the 1983 extracts over time).

Data from all the samples tested as well as multiple lots obtained from a single company are summarized in Fig 3. The mean Asp f 1 level was 22.4 ± 22.6 µg/mL (n = 20, those containing >0.2 µg/mL), with extracts

644 Vailes et al

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Spore extracts Because the major route of Alternaria exposure is through inhalation of spores, we evaluated Alt a 1 content in extracts made from both dried and actively growing Alternaria spores. Alt a 1 was detected at concentrations of 11 to 73 ng/mL (Table III).

DISCUSSION

FIG 3. Comparison of major allergen levels in fungal extracts. Cumulative data for Asp f 1 levels in A fumigatus (left column) or Alt a 1 in Alternaria (right column) for US commercial extracts, 1998/1999 (); production lots from a single manufacturer (■); or extracts/standard prepared in 1983 (▲). Mean levels are indicated by the horizontal bar.

TABLE II. Alt a 1 concentrations of extracts used 17 years ago to prepare an Alternaria reference* Manufacturer

Greer Stallergenes Pharmacia Bayer Riem Bulgaria Proposed International Biological Reference

Preparation date

6/83 7/83 5/83 7/83 7/83 1983

Alt a 1 (µg/mL)

1.63 1.23 1.07 1.00 <0.04 0.79

*Lyophilized material stored at –20°C in glass ampules.

TABLE III. Alt a 1 levels in spores Source material

Dry spores UNLV spores/hyphae in solution UNLV spores/hyphae in solution

Spore concentration

Alt a 1 (ng/mL)

56 mg/mL 5 × 105 mL

64 11

1.5 × 105 mL

73

UNLV, University of Nevada, Las Vegas.

showing almost a 100-fold variation in Asp f 1 content. The mean for Alternaria was 1.4 ± 1.1 µg/mL Alt a 1 (n = 27, those containing >0.2 µg/mL), and 21 of 32 (66%) of these extracts contained 0.7 to 2.0 µg/mL Alt a 1. Data for multiple extracts prepared by the same company closely mirrored the results found for the 8 individual companies. Intracompany results for Alt a 1 were 1.2 ± 0.3 µg/mL (n = 10, range 0.49-1.42 µg/mL) and 17.0 ± 19.8 µg/mL for Asp f 1 (n = 12, range 0.96-55.11 µg/mL).

The results show that the Alt a 1 ELISA using rAlt a 1 as a standard is a useful tool for analyzing Alt a 1 levels in natural allergenic products. The assay is specific for Alternaria and is highly sensitive (down to 0.2 ng/mL Alt a 1). A somewhat surprising result was that the recombinant allergen, produced as a 15-kd protein, gave equivalent ELISA results to natural Alt a 1, which exists as a 29-kd dimer. This is most likely explained by the ability of rAlt a 1 produced in bacteria to spontaneously dimerize, which has also been reported for rAlt a 1 produced in Pichia pastoris.30 The advantage of using rAlt a 1 as a standard is that unlike natural allergen, it can be produced by bacteria (or yeast) in essentially unlimited supplies and is accurately quantitated. Although it has generally been thought that Alternaria extracts varied significantly in allergen content, the data show that production methods used by different manufacturers result in extracts that contain relatively consistent Alt a 1 levels (~1 µg/mL). This is remarkable for unstandardized extracts labeled as 1:10 or 1:20 wt/vol and was consistent both between manufacturers and over time (1983, 1998, 1999). Similar batch-to-batch consistency was reported for Alternaria extracts from a European manufacturer (Allergopharma).31 The absolute values for US allergenic products reported here are 2- to 14-fold lower than those recently reported with another ELISA for Alt a 1.33 The previous assay used 2 mAbs and reported 12 to 84 µg/mL Alt a 1 in 3 Alternaria extracts. The most likely explanation for these discrepancies is the use of different Alt a 1 standards in the assays. The availability of rAlt a 1 for use as a standard should resolve these differences. In comparison with results of Alternaria products, results of Asp f 1 levels in the Aspergillus extracts were quite variable both between manufacturers and between different lots from the same company. The scale of these differences (Asp f 1 undetectable vs 4-65 µg/mL) almost certainly means that the manufacturers used different methods for producing Aspergillus extract. Asp f 1 is secreted at high concentrations in A fumigatus culture filtrate but is present at 1000-fold lower levels in spores or disrupted hyphae.14 Conversely, Alt a 1 is present in both spores and mycelium and appears to be extracted more uniformly.34 The results raise questions about whether there are significant quantitative differences between other A fumigatus allergens in commercial extracts and whether these are likely to have clinical significance. The strong association of Alternaria sensitivity as a risk factor for asthma in the United States, Australia, and in parts of France prompted us to investigate whether the

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Alt a 1 ELISA could be used to assess environmental exposure to Alternaria.6,7,9-11,35 The fact that Alt a 1 was detected in both an extract made from 20-year-old dried spores and spore suspensions taken from actively growing Alternaria shows that Alt a 1 is present in spores. However, detection of Alt a 1 in the ELISA required high spore concentrations (105 to 106/mL), which is probably much greater than that likely to be found in most house dust or air samples. In fact, we assayed 1531 dust extracts from the National Cooperative Inner City Asthma Study and others in which Alternaria sensitivity was high, and we detected Alt a 1 in only 6 samples (data not shown). These observations suggest that although the Alt a 1 ELISA is very sensitive (0.2 ng/mL), high levels of environmental fungal exposure would be needed to be detected in this assay. In 1997 the American Academy of Allergy, Asthma and Immunology published a position statement advocating the use of standardized allergen extracts based on skin test potency and measurement of major allergen content.15 It is not clear whether a concentration of 1 µg/mL Alt a 1 is optimal for diagnostic efficacy on skin testing, and further studies would be required to determine whether this level is suitable for standardization. However, Aden et al31 found that potency of Alternaria allergen correlated with skin test reactivity, and 12 of 13 patients had positive skin prick test responses to less than 1 µg/mL Alt a 1. Using Center for Biologic Evaluation and Research reference preparations or recombinant allergens, manufacturers may use ELISA testing as a means of standardization and for control of lot consistency of allergen extracts. Currently, cat, Dermatophagoides pteronyssinus, Dermatophagoides farinae, ragweed, grass pollens, and some venoms are the only standardized allergen extracts approved by the Food and Drug Administration. The results of this study show that the specific mAb-based ELISAs for Alt a 1 and Asp f 1 will serve as useful tools in the standardization and quality control of Alternaria and Aspergillus allergenic products. In addition, because rAlt a 1 bound equally as well as natural Alt a 1 in the ELISA, it is ideal for use as a purified reference standard of known concentration. We greatly appreciate the help of several colleagues who contributed reagents and samples for these studies: Drs Greg Plunkett, John Yunginger, and Visnavath Kurup (fungal allergen extracts); and Drs Don Hoffman and Linda Stetzenbach (Alternaria spores). We are also grateful to Wanda Harvey for administrative assistance.

REFERENCES 1. Al-Doory Y, Ramsey S. Mould and health, who is at risk. Springfield (IL): Charles C Thomas; 1987. 2. Aukrust L, Borch SM, Einarsson R. Mould allergy: spores and mycelium as allergy sources. Allergy 1985;40:43-8. 3. Platts-Mills TAE, Solomon WR. Aerobiology and inhalant allergens. In: Middleton E, Reed CE, Ellis EF, Adkinson NF, Yunginger JW, Busse WW, eds. Allergy principles and practice. 4th ed. St Louis: Mosby; 1993. p. 498-514. 4. Henderson FW, Henry MM, Ivins SS, Morris R, Neebe EC, Leu SY, et al. Correlates of recurrent wheezing in school age children. Am J Respir Crit Care Med 1995;151:1786-93.

Vailes et al 645

5. Tariq SM, Matthews SM, Stevens M, Hakim EA. Sensitization to Alternaria and Cladosporium by the age of 4 years. Clin Exp Allergy 1996;26:794-8. 6. Gergen PJ, Turkeltaub PC. The association of individual allergen reactivity and respiratory disease in a national sample: data from the Second National Health and Nutrition Examination Survey, 1976-80 (NHANES II). J Allergy Clin Immunol 1992;90:579-88. 7. Perzanowski MS, Sporik R, Squillace SP, Gelber LE, Call R, Carter M, et al. Association of sensitization to Alternaria allergens with asthma among school-age children. J Allergy Clin Immunol 1998;101:626-32. 8. Nelson HS, Szefler SJ, Jacobs J, Huss K, Shapiro G, Sternberg AL. The relationships among environmental allergen sensitization, allergen exposure, pulmonary function, and bronchial hyperresponsiveness in the Childhood Asthma Management Program. J Allergy Clin Immunol 1999;104:775-85. 9. Peat JK, Woolcock AJ. Sensitivity to common allergens: relation to respiratory symptoms and bronchial hyper responsiveness in children from three different climatic areas of Australia. Clin Exp Allergy 1991;21:573-81. 10. Peat JK, Tovey E, Mellis CM, Leeder SR, Woolcock AJ. Importance of house dust mite and Alternaria allergens in childhood asthma: an epidemiological study in two climatic regions of Australia. Clin Exp Allergy 1993;23:812-20. 11. Halonen M, Stern DA, Wright AL, Taussig LM, Martinez FD. Alternaria as a major allergen for asthma in children raised in a desert environment. Am J Respir Crit Care Med 1997;155:1356-61. 12. O’Hollaren MT, Yunginger JW, Offord KP, Somers MJ, O’Connell EJ, Ballard DJ, et al. Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma. N Engl J Med 1991;324:359-63. 13. Crameri R. Recombinant Aspergillus fumigatus allergens: from the nucleotide sequence to clinical applications. Int Arch Allergy Immunol 1998;115:99-114. 14. Arruda LK, Man BJ, Chapman MD. Selective expression of a major allergen and cytotoxin, Asp f 1, in Aspergillus fumigatus. Implications for the immunopathogenesis of Aspergillus related diseases. J Immunol 1992;149:3354-9. 15. AAAAI. Position statement. The use of standardized allergen extracts. J Allergy Clin Immunol 1997;99:583-6. 16. Vijay HM, Huang H, Young NM, Bernstein IL. Studies on Alternaria allergens. IV. Comparative biochemical and immunological studies of commercial Alternaria tenuis batches. Int Arch Allergy Appl Immunol 1984;74:256-61. 17. Steringer I, Aukrust L, Einarsson R. Variability of antigenicity/allergenicity in different strains of Alternaria alternata. Int Arch Allergy Appl Immunol 1987;84:190-7. 18. Portnoy J, Pacheco F, Ballam Y, Barnes C. The effect of time and extraction buffers on residual protein and allergen content of extracts derived from four strains of Alternaria. J Allergy Clin Immunol 1993;91:930-8. 19. Paris S, Fitting C, Ramirez E, Latgé DB. Comparison of different extraction methods of Alternaria allergens. J Allergy Clin Immunol 1990;85:941-8. 20. Wahl R, Oliver JD, Hauck PR, Winter HG, Maasch HJ. Comparison of Alternaria extracts prepared from different raw materials. Ann Allergy 1989;63:527-31. 21. Van Der Heide S, Kauffman HF, De Vries K. Cultivation of fungi in synthetic and semi synthetic liquid medium. II. Immunochemical properties of antigenic and allergenic extracts. Allergy 1985;40:592-8. 22. Agarwal MK, Jones RT, Yunginger JW. Immunochemical and physicochemical characterization of commercial Alternaria extracts: a model for standardization of mold allergen extracts. J Allergy Clin Immunol 1982;70:432-6. 23. Helm RM, Squillace DL, Yunginger JW, Members of the International Collaborative Trial. Production of a proposed international reference standard Alternaria extract. II. Results of collaborative trial. J Allergy Clin Immunol 1988;81:651-63. 24. Yunginger JW, Jones RT, Nesheim ME, Geller M. Studies on Alternaria allergens. III. Isolation of a major allergenic fraction (ALT-I). J Allergy Clin Immunol 1980;66:138-47. 25. Kleine-Tebbe J, Worm M, Jeep S, Matthiesen F, Lowenstein H, Kunkel G. Predominance of major allergen (Alt a 1) in Alternaria sensitized patients. Clin Exp Allergy 1993;23:211-18. 26. Curran IHA, Young NM, Burton M, Vijay HM. Purification and characterization of Alt a 29 from Alternaria alternata. Int Arch Allergy Immunol 1993;102:267-75.

646 Vailes et al

27. Achatz G, Oberkofler H, Lechenauer E, Simon B, Unger A, Kandler D, et al. Molecular cloning of major and minor allergens of Alternaria alternata and Cladosporium herbarium. Mol Immunol 1995;32:213-27. 28. Vijay HM, Young NM, Curran IHA, Copeland DF, Bernstein IL. A major antigen of Alternaria alternata with potential for safe and effective immunotherapy. J Allergy Clin Immunol 1993;91:826-8. 29. Zhang L, Curran IHA, Muradia G, De Vouge MW, Rode H, Vijay H. Nterminus of a major allergen, Alt a 1, of Alternaria alternata defined to be an epitope. Int Arch Allergy Immunol 1995;108:254-9. 30. DeVouge MW, Thaker AJ, Curren IHA, Zhang L, Muradia G, Rode H, et al. Isolation and expression of a cDNA clone encoding an Alternaria alternata Alt a 1 subunit. Int Arch Allergy Immunol 1996;111:385-95. 31. Aden E, Weber B, Bossert J, Wahl R, Teppke M, Frank E, et al. Standardization of Alternaria alternata: extraction and quantification of Alt a 1 by using an mAb-based binding assay. J Allergy Clin Immunol 1999;103:128-35.

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32. Ovsyannikova IG, Vailes LD, Li Y, Heymann PW, Chapman MD. Monoclonal antibodies to group II Dermatophagoides spp. allergens: murine immune response, epitope analysis, and development of a two-site ELISA. J Allergy Clin Immunol 1994;94:537-46. 33. Portnoy J, Brothers D, Pacheco F, Landuyt J, Barnes C. A monoclonal antibody-based assay for Alt a 1, a major Alternaria allergen. Ann Allergy Asthma Immunol 1998;81:59-64. 34. Paris SC, Fitting C, Ramirez E, Latge JP, Herman D, Gulnnepain MT, et al. Comparison of conidial and mycelial allergens of Alternaria alternata. Int Arch Appl Immunol 1990;92:1-8. 35. Neukirch C, Henry C, Leynaert B, Liard R, Bousquet J, Neukirch F. Is sensitization to A alternata a risk factor for severe asthma? A populationbased study. J Allergy Clin Immunol 1999;103:709-11.