Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota).

Abstract

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosis.

• Authors: Kay Foetisch, Lotte Dahl, Baerbel Jansen, Wolf-Meinhard Becker, Jonas Lidholm, Ronald van Ree, Hermann Broll, Susanne Kaul, Stefan Vieths, Thomas Holzhauser
• Journal: Analytical and bioanalytical chemistry (Anal Bioanal Chem) Vol. 399 Issue 2 Pg. 935-43 (Jan 2011) ISSN: 1618-2650 [Electronic] Germany
• PMID: 21046078 (Publication Type: Journal Article, Validation Study)

Chemical References

• Allergens
• Antibodies, Monoclonal
• Plant Extracts
• Plant Proteins
• Protein Isoforms

Topics

• Allergens (analysis, immunology)
• Animals
• Antibodies, Monoclonal (analysis, immunology)
• Daucus carota (chemistry, immunology)
• Enzyme-Linked Immunosorbent Assay (methods)
• Female
• Limit of Detection
• Mice
• Mice, Inbred BALB C
• Plant Extracts (chemistry, immunology)
• Plant Proteins (analysis, immunology)
• Protein Isoforms (analysis, immunology)