Physico-chemical features of the environment affect the protein conformation and the immunoglobulin E reactivity of kiwellin (Act d 5).
BACKGROUND
Allergy diagnostic systems sometimes give false positive or negative results. In this respect, the influence of protein conformational changes on the allergen-IgE interaction sites is worthy to be investigated.


OBJECTIVE
To investigate the influence of different experimental conditions on the structural properties and IgE reactivity of kiwellin (Act d 5) as a model system.


METHODS
Act d 5 was purified from the natural source. To study its conformational features, experiments of circular dichroism (CD) in different media were performed. The IgE reactivity was investigated by skin testing, immunoblotting and ISAC microarray system, in a population of kiwifruit allergic subjects.


RESULTS
CD experiments indicated that Act d 5 has a mainly helical structure and the conformation is strongly affected by the experimental conditions. The protein is more structured in low polarity media and at acidic pH values, similar to those of the natural source. Eleven subjects of 29 (38%) allergic to kiwifruit were positive to purified natural Act d 5 by skin test. Among them, three patients (10%) showed a reaction only to Act d 5 at pH 4.5, and three (10%) showed a reaction only to the allergen in standard neutral conditions. No one of the 11 subjects with positive skin test recognized Act d 5 immobilized on the ISAC system. Eight of nine subjects detected Act d 5 by IgE immunoblotting. One subject did not recognize the sequence epitopes of Act d 5 in IgE immunoblotting experiments and reacted to the skin test only when the allergen was in acidic conditions.


CONCLUSIONS
The conformation and IgE reactivity of Act d 5 are affected by the physico-chemical characteristics of the solvent. These findings suggest that the assay conditions influence the results of the diagnostic systems by modulating the pattern of exposed antigenic epitopes.
Bernardi
M L
ML
Center for Molecular Allergology, IDI-IRCCS, Roma, Italy.
Picone
D
D
Tuppo
L
L
Giangrieco
I
I
Petrella
G
G
Palazzo
P
P
Ferrara
R
R
Tamburrini
M
M
Mari
A
A
Ciardiello
M A
MA
D029042:Actinidia / Q000276:immunology*; D000293:Adolescent; D000328:Adult; D052179:Antigens, Plant / Q000737:chemistry* / Q000276:immunology*; D002942:Circular Dichroism; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D005260:Female; D005512:Food Hypersensitivity / Q000175:diagnosis*; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D008297:Male; D008875:Middle Aged; D017433:Protein Structure, Secondary; D012882:Skin Tests; D012997:Solvents / Q000737:chemistry; D055815:Young Adult
D052179:Antigens, Plant; D000939:Epitopes; D012997:Solvents; C507612:kiwellin protein, Actinidia chinensis; D007073:Immunoglobulin E