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.
Yi
F C
FC
Departments of Paediatrics and Otolaryngology, Faculty of Medicine and Bioprocessing Technology Centre, National University of Singapore, Republic of Singapore.
Cheong
N
N
Shek
L P
LP
Shek
P C L
PC
Wang
D Y
DY
Chua
K Y
KY
Lee
B W
BW
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