Peptide mapping of immunoglobulin E and immunoglobulin G immunodominant epitopes of an allergenic Blomia tropicalis paramyosin, Blo t 11.
BACKGROUND
The identification of immunodominant peptides containing the IgE and IgG epitopes on allergen molecules is an important step in understanding the interaction of the allergen with the immune system and, thus, essential for the development of effective immunotherapeutic and diagnostic reagents. The present study aimed to map the IgE and IgG immunodominant peptides of Blomia tropicalis (Bt) allergen Blo t 11, a high molecular weight allergen homologous to paramyosin, exhibiting important allergenic activity.


METHODS
Eleven overlapping fragments of Blo t 11 cDNA gene were expressed as glutathione s-transferase (GST) fusion peptides, which were affinity-purified using the glutathione-Sepharose column. Human IgE and IgG immunodominant peptides were determined by dot blot immunoassay using crude Bt extract-positive sera from asthmatic patients. Evaluation of allergenicity, specific hIgG subclass analysis, and cross- and self-inhibition studies were determined by enzyme-linked immunosorbent assay.


RESULTS
Blo t 11 contains multiple IgE and IgG immunodominant peptides scattered throughout the molecule. The dominant IgE and IgG peptides were mapped at amino acid positions 336-557 and 698-875, respectively. An immunodominant peptide (fD) registered a higher percentage of IgE and IgG reactivity compared to the rFL-Blo t 11. Significant serum levels of Blo t 11- and fD-specific IgG1, IgG2 and IgG4, but not IgG3 were detected in the Bt extract-positive sera tested. Cross-inhibition study revealed the rFL-Blo t 11 was significantly inhibited by fD.


CONCLUSIONS
The IgE and IgG immunodominant peptides of Blo t 11 have been mapped. Our data suggest that utilization of Blo t 11 fragment(s) or chimeric fusion fragments containing IgE and IgG epitopes could be a better alternative in the development of diagnostic and therapeutic reagents for mite allergy.
Ramos
J D A
JD
Department of Paediatrics, Faculty of Medicine, National University of Singapore, Singapore.
Cheong
N
N
Lee
B W
BW
Chua
K Y
KY
D000485:Allergens / Q000737:chemistry*; D000818:Animals; D052179:Antigens, Plant; D001249:Asthma / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D008925:Mites; D010449:Peptide Mapping
D000485:Allergens; D052179:Antigens, Plant; C448821:Blo t 11 allergen; D000939:Epitopes; D007074:Immunoglobulin G; D007073:Immunoglobulin E