Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus.
BACKGROUND
Tropomyosin belongs to a class of highly conserved proteins in invertebrates and vertebrates. The invertebrate tropomyosins are allergenic in man with high IgE cross-reactivity and have been therefore referred to as pan-allergens.


OBJECTIVE
This study aimed to clone and identify the IgE epitopes of tropomyosin from Blomia tropicalis (Blo t 10) mite. Cross-reactivity between the IgE epitopes of Blo t 10 and Der p 10 was also evaluated.


METHODS
Blo t 10 was isolated using mouse anti-Der p 10 antibodies. Allergenicity of the cloned Blo t 10 was confirmed by skin prick test (SPT) and enzyme-linked immunosorbent assay (ELISA). Dose-dependent inhibition assay was performed to determine the degree of IgE cross-reactivity between Blo t 10 and Der p 10. Overlapping polymerase chain reaction-derived cDNA were generated and expressed as glutathione-S-transferase (GST) recombinant proteins in Escherichia coli and used to identify shared and unique IgE epitopes of Blo t 10 and Der p 10.


RESULTS
The cloned Blo t 10 shared up to 96% amino acid identity to tropomyosin of other mites. SPT and ELISA IgE-immunoassay showed recombinant Blo t 10 sensitization rates of between 20% and 29% in atopic subjects. Results of SPT and dose-dependent inhibition assays showed that some allergic individuals had unique IgE epitopes for Blo t 10. IgE epitope mapping of Blo t 10 revealed that the epitopes were mainly located at N- and C-termini of the molecule. The results of ELISA inhibition assays of overlapping recombinant fragments indicated that the unique IgE epitopes of Blo t 10 were located at the C-terminal.


CONCLUSIONS
Although Blo t 10 and Der p 10 are highly conserved (shared 95% amino acids identity) and significantly cross-reactive, unique IgE epitopes do exist. The results suggest the potential deficiency of using only one of these highly conserved allergens as diagnostic or therapeutic reagents.
Departments of Paediatrics and Otolaryngology, Faculty of Medicine and Bioprocessing Technology Centre, National University of Singapore, Republic of Singapore.
F C
FC
Yi
N
N
Cheong
L P
LP
Shek
P C L
PC
Shek
D Y
DY
Wang
K Y
KY
Chua
B W
BW
Lee
D000293:Adolescent; D000328:Adult; D000368:Aged; D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002648:Child; D002675:Child, Preschool; D003429:Cross Reactions; D015342:DNA Probes; D040002:Dermatophagoides pteronyssinus / Q000276:immunology; D004391:Dust; D000939:Epitopes / Q000032:analysis* / Q000235:genetics; D015723:Gene Library; D006801:Humans; D006967:Hypersensitivity / Q000276:immunology*; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology*; D051379:Mice; D008875:Middle Aged; D008925:Mites / Q000276:immunology*; D017422:Sequence Analysis, DNA; D012846:Singapore; D012882:Skin Tests; D014335:Tropomyosin / Q000276:immunology*
D000485:Allergens; D015342:DNA Probes; D004391:Dust; D000939:Epitopes; D014335:Tropomyosin; D007073:Immunoglobulin E