Mapping of Dermatophagoides farinae mite allergens by two-dimensional immunoblotting.
BACKGROUND
Allergens from the house dust mite Dermatophagoides farinae are responsible for frequent respiratory allergic disorders, but only 3 groups of these allergens are well characterized.


OBJECTIVE
This study was performed to complete the repertoire of D farinae allergens using two-dimensional (2-D) electrophoresis.


METHODS
D farinae mite allergens, extracted from whole cultures in the presence of a mild detergent, were separated by 2-D electrophoresis with subsequent immunoblotting. IgE-binding proteins were detected with individual mite-sensitive patient sera and the anti-D pteronyssinus human serum pool. Allergens were identified by an inhibition immunoblot test, by means of specific mAbs, or by biochemical characterization. The internal peptides of 2 allergens were microsequenced.


RESULTS
2-D immunoblotting with individual patient sera showed a marked heterogeneity in the isoelectric point of the allergens, as well as differences in the individual IgE-binding patterns. In addition to identification of allergens Der f 1, Der f 2, and Der f 3, new allergens have been characterized as Der f 4, Der f 5, and 2 high molecular mass allergens. Microsequencing of peptides from the latter allergens revealed significant homologies with allergen Mag 3 from D farinae and with a chitinase from prawn Penaeus japonicus.


CONCLUSIONS
2-D electrophoresis with subsequent immunoblotting and protein microsequencing allowed characterization of a more complete repertoire of D farinae allergens and their multiple isoforms, and identification of six new allergens.
Le Mao
J
J
Unité d'Immuno-Allergie, Institut Pasteur, Paris, France.
Mayer
C E
CE
Peltre
G
G
Desvaux
F X
FX
David
B
B
Weyer
A
A
Sénéchal
H
H
D000485:Allergens / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D039741:Antigens, Dermatophagoides; D002688:Chitinases / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D008925:Mites / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping
D000485:Allergens; D039741:Antigens, Dermatophagoides; D006023:Glycoproteins; D002688:Chitinases