Standardization of Alternaria alternata: extraction and quantification of alt a 1 by using an mAb-based 2-site binding assay.
BACKGROUND
Alternaria alternata is recognized as an important cause of allergic disease. As with other molds, the extracts of A alternata used for diagnosis and therapy are highly heterogeneous, and there is a need for improved standardization. The major allergen Alt a 1 is well characterized and has been produced as a recombinant protein, but very few data are available on the Alt a 1 content in extracts.


OBJECTIVE
An assay for the quantification of Alt a 1 was developed and used for monitoring batch-to-batch consistency of A alternata extracts, and the correlation between skin prick test responses and Alt a 1 concentrations was studied.


METHODS
A 2-site binding assay based on an Alt a 1-specific mAb was developed and used for the quantification of Alt a 1 in allergen extracts. Quantitative skin prick tests were performed on 16 A alternata-sensitive patients and correlated with the Alt a 1 concentration.


RESULTS
The Alt a 1-specific mAb was found to be suitable for affinity purification, as well as for a 2-site binding quantification assay. In allergen extracts the Alt a 1 content was estimated as 2% to 4.7% of total protein. Quantitative skin prick tests showed an Alt a 1 concentration-dependent response.


CONCLUSIONS
Quantification of Alt a 1 in A alternata extracts reflects their batch-to-batch consistency. Skin prick test responses to a standardized A alternata extract correlate with the Alt a 1 contents. An extract containing 3.7 microgram/mL Alt a 1 caused a response equal to that of a 1% histamine dihydrochloride solution.
Aden
E
E
Allergopharma Joachim Ganzer KG, Reinbek; Mannheim; the Department of Biochemistry and Molecular Biology, University of Hamburg, Germany.
Weber
B
B
Bossert
J
J
Teppke
M
M
Frank
E
E
Wahl
R
R
Fiebig
H
H
Cromwell
O
O
D000485:Allergens / Q000276:immunology; D000528:Alternaria; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D052179:Antigens, Plant; D001666:Binding Sites, Antibody; D005656:Fungal Proteins / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D010936:Plant Extracts / Q000592:standards; D011817:Rabbits; D012882:Skin Tests
C096258:ALTA1 protein, Alternaria alternata; D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D005656:Fungal Proteins; D010936:Plant Extracts