A sensitive two-site enzyme-linked immunosorbent assay for measurement of the major Alternaria alternata allergen Alt a 1

Background: Alt a 1 is the major allergen in Alternaria alternata, one of the most important fungi associated with allergic diseases. Mold allergenic extracts show considerable heterogeneity, and thus accurate standardization of these extracts is essential to guarantee their quality. Objective: To develop an Alt a 1-specific assay and to evaluate the correlation of Alt a 1 content with the IgE-binding activity of A. alternata extracts. Methods: Recombinant Alt a 1 was produced as nonfusion protein from a polymerase chain reaction-cloned complementary DNA Alt a 1 sequence. Natural Alt a 1 was purified from spent culture medium. Monoclonal and polyclonal antibodies directed to Alt a 1 were produced and used to construct a specific Alt a 1 enzyme-linked immunosorbent assay (ELISA). Results: The ELISA developed was highly reproducible and sensitive, with a detection limit lower than 0.5 ng/mL and a practical working range of 0.5 to 50 ng/mL. The assay was able to detect an Alt a 1-like protein in Stemphylium extracts. Identical parallel dose-response curves were observed when natural Alt a 1, and recombinant Alt a 1 were used as standard. A good correlation was obtained between Alt a 1 content of 13 A. alternata extracts and their IgE-binding activity. Alt a 1 was responsible for 70% of the IgE-binding activity of the whole extract. Conclusions: This sensitive and specific Alt a 1 assay allows the quantification of this major mold allergen and represents a useful tool for the standardization of A. alternata extracts in mass units. It also provides a reliable indication of the allergenic activity of the whole extract.