Background: Cypress pollen allergy is an important cause of rhinoconjunctivitis and asthma in Mediterranean countries. Cypress allergenic extracts are difficult to produce since they have low protein and high carbohydrate content, thus accurate standardization of them is essential to guarantee their quality. The aim of this study is to develop a sandwich ELISA for the quantification of Cup a 1, the major allergen of cypress (Cupressus arizonica) pollen extract. Methods: Monoclonal antibodies directed to purified Cup a 1 were produced. Two of them (9C7 as capture antibody and 3D2 as the tracer) were selected to develop a quantitative sandwich ELISA. This ELISA was subsequently evaluated and compared with other techniques. Results: The described ELISA is very sensitive with a detection limit of 8.7 ng/ml and a practical working range of 62.5–1,000 ng/ml. The assay is also highly reproducible with intra-assay and interassay coefficients of variation of less than 10%. The purified Cup a 1, used as standard, presents pectate lyase enzymatic activity. The assay also detected Cup a 1-like proteins in pollen from other Cupressaceae. A good correlation was obtained between Cup a 1 content of 12 C. arizonica pollen extracts and their IgE-binding activity. Conclusions: The described Cup a 1 ELISA is sensitive, specific and reproducible and can be used for the quantification of Cup a 1 in C. arizonica and other related pollen extracts. It also provides a reliable indication of the allergenic activity of the whole cypress pollen extract.

Keywords:
Allergen standardization, Cross-reactivity, Cypress pollen, Immunoassay, Monoclonal antibodies
1.
Panzani R, Centanni G, Brunel M: Increase of respiratory allergy to the pollens of cypresses in the south of France. Ann Allergy 1986;56:460–463.
2.
Bousquet J, Cour P, Guerin B, Michel FB: Allergy in the Mediterranean area. I. Pollen counts and pollinosis of Montpellier. Clin Allergy 1984;14:249–258.
3.
Caballero T, Romualdo L, Crespo JF, Pascual C, Munoz-Pereira M, Martín-Esteban M: Cupressaceae pollinosis in the Madrid area. Clin Exp Allergy 1996;26:197–201.
4.
Dubus JC, Melluso JP, Bodiou AC, Stremler-Lebel N: Allergy to cypress pollen. Allergy 2000;55:418–419.
5.
Mari A, Di Felice G, Afferni C, Barletta B, Tinghino R, Pini C: Cypress allergy: An underestimated pollinosis. Allergy 1999;52:355–356.
6.
Di Felice G, Caiaffa MF, Bariletto G, Afferni C, Di Paola R, Mari A, Palumbo S, Tinghino R, Sallusto F, Tursi A, Macchia L, Pini C: Allergens of Arizona cypress (Cupressus arizonica) pollen: Characterization of the pollen extract and identification of the allergenic components. J Allergy Clin Immunol 1994;94:547–555.
7.
Ford SA, Baldo BA, Panzani R, Bass D: Cypress (Cupressus sempervirens) pollen allergens: Identification by protein blotting and improved detection of specific IgE antibodies. Int Arch Allergy Appl Immunol 1991;95:178–183.
8.
Alisi C, Afferni C, Iacovacci P, Barletta B, Tinghino R, Butteroni C, Puggioni EMR, Wilson IBH, Federico R, Schinina ME, Ariano R, Di Felice G, Pini C: Rapid isolation, characterization, and glycan analysis of Cup a 1, the major allergen of Arizona cypress (Cupressus arizonica) pollen. Allergy 2001;56:978–984.
9.
Di Felice G, Barletta B, Tinghino R, Pini C: Cupressaceae pollinosis: Identification, purification and cloning of relevant allergens. Int Arch Allergy Immunol 2001;125:280–289.
10.
Aceituno E, Del Pozo V, Mínguez A, Arrieta I, Cortegano I, Cárdaba B, Gallardo S, Rojo M, Palomino P, Lahoz C: Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1. Clin Exp Allergy 2000;30:1750–1758.
11.
Bousquet J, Lockey R, Malling HJ: Allergen immunotherapy: Therapeutic vaccines for allergic diseases. Allergy 1998;53:1–42.
12.
Keen N, Dahlbeck D, Staskawics BJ, Belser W: Molecular cloning of pectate lyase genes from Erwinia chrysanthemi and their expression in Escherichia coli. J Bacteriol 1984;159:825–831.
13.
Arilla MC, Asturias JA, Gómez-Bayón N, Martínez A, Martínez J, Palacios R: Production and characterization of profilin monoclonal antibodies. Allergol Immunopathol (Madr) 1997;25:145–151.
14.
Arilla MC, Ibarrola I, Eraso E, Aguirre M, Martínez A, Asturias JA: Quantification in mass units of group 1 grass allergens by a monoclonal antibody-based sandwich ELISA. Clin Exp Allergy 2001;31:1271–1278.
15.
Woodward MP, Young WW, Bloodgood RA: Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation. J Immunol Methods 1985;78:143–153.
16.
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;277:680–685.
17.
Arilla MC, Eraso E, Ibarrola I, Algorta J, Martínez A, Asturias JA: Monoclonal antibody-based method for measuring Ole e 1 olive pollen allergen. Ann Allergy Asthma Immunol 2002;89:83–89.
18.
Towbin H, Staehelin I, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 1979;76:4350–4354.
19.
Bousquet J, Knani J, Hejjaoui A, Ferrando R, Cour P, Dhivert H, Michel FB: Heterogeneity of atopy. I. Clinical and immunologic characteristics of patients allergic to cypress pollen. Allergy 1993;48:183–188.
20.
Mari A, Di Felice G, Afferni C, Barletta B, Tinghino R, Sallusto F, Pini C: Assessment of skin prick test and serum specific IgE detection in the diagnosis of Cupressacea pollinosis. J Allergy Clin Immunol 1996;98:21–31.
21.
Hrabina M, Dumur J-P, Sicard H, Viatte A, André C: Diagnosis of cypress pollen allergy: In vivo and in vitro standardization of a Juniperus ashei pollen extract. Allergy 2003;58:808–813.
22.
Iacovacci P, Afferni C, Butteroni C, Pironi L, Puggioni EMR, Orlandi A, Barletta B, Tinghino R, Ariano R, Panzani RC, Di Felice G, Pini C: Comparison between the native glycosylated and the recombinant Cup a 1 allergen: Role of carbohydrates in the histamine release from basophils. Clin Exp Allergy 2002;32:1620–1627.
23.
Lahoz C, Cortegano I, Mínguez A, Del Pozo V, Civantos E, Cárdaba B, Aceituno E, Rojo M, Gallardo S, Palomino P: Cup a 3, major allergen from Cupressus arizonica: Cloning and expression in Pichia pastoris. J Allergy Clin Immunol 2002;109:136–137.
24.
van Ree R: A new start for allergen references and standardization based on purified (natural or recombinant) allergens and monoclonal and monospecific polyclonal antibodies; in Kurth R, Haustein D (eds): Regulatory Control and Stardardization of Allergenic Extracts. Darmstadt, GIT Verlag, 2000, pp 87–92.
25.
Asturias JA, Arilla MC, Ibarrola I, Eraso E, González-Rioja R, Martínez A: A sensitive two-site enzyme-linked immunosorbent assay for measurement of the major Alternaria alternata allergen Alt a1. Ann Allergy Asthma Immunol 2003;90:529–535.
26.
González R, Duffort O, Calabozo B, Barber D, Carreira J, Polo F: Monoclonal antibody-based method to quantify Gly m 1. Its application to assess environmental exposure to soybean dust. Allergy 2000;55:59–64.
27.
Fahlbusch B, Schlenvoigt G, Müller WD, Weber B, Ja ger L: A two-site monoclonal antibody ELISA for the quantification of group V allergens in grass extracts. Clin Exp Allergy 1994;24:752–757.
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2004
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