Comparative allergenicity studies of native and recombinant Blomia tropicalis Paramyosin (Blo t 11).
BACKGROUND
The complementary DNA (cDNA) encoding for Blo t 11, a 102 kD allergen from Blomia tropicalis (Bt) was isolated, expressed and characterized previously. This study aimed to isolate the native Blo t 11 allergen and compare its allergenicity with the recombinant forms.


METHODS
Native Blo t 11 (nBlo t 11) was isolated from crude Bt extract by immuno-affinity chromatography, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and verified by MALDI-TOF MS. Recombinant full-length Blo t 11 (rFL-Blo t 11) and its immunodominant peptide (fD) were expressed as glutathione S-transferase (GST)-fusion proteins in Escherichia coli. Immunoglobulin E (IgE) reactivity of the Blo t 11 allergens were determined by enzyme-linked immunosorbent assay (ELISA) and skin prick test. The inhibition capacity of the nBlo t 11 against fD and vice versa was determined by absorption studies.


RESULTS
Affinity purified nBlo t 11 was susceptible to degradation with the major degraded product resolved at approximately 66 kD. The nBlo t 11 was confirmed by immunoblot analysis and MALDI-TOF MS that generated 13 peptides with complete identity to the deduced amino acid sequence of Blo t 11. Comparative in vitro and in vivo allergenicity tests and the cross inhibition studies between the native and recombinant Blo t 11 showed that recombinant fD, but not the rFL-Blo t 11, has comparable IgE reactivity with the native counterpart.


CONCLUSIONS
This comparative study confirmed that the recombinant peptide fD contains the main immunodominant region of Blo t 11. This recombinant peptide, instead of the full-length protein, is a good candidate for diagnostic and therapeutics development for mite allergy.
Ramos
J D A
JD
Department of Paediatrics, Faculty of Medicine, National University of Singapore, Singapore.
Teo
A S M
AS
Ou
K L
KL
Tsai
L C
LC
Lee
B W
BW
Cheong
N
N
Chua
K Y
KY
D000485:Allergens / Q000276:immunology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D052179:Antigens, Plant; D001249:Asthma / Q000276:immunology*; D016022:Case-Control Studies; D003429:Cross Reactions / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000097:blood; D008925:Mites / Q000276:immunology; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000276:immunology*; D012882:Skin Tests; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C448821:Blo t 11 allergen; D011994:Recombinant Proteins; D007073:Immunoglobulin E