DNA immunization for the production of monoclonal antibodies to Blo t 11, a paramyosin homolog from Blomia tropicalis.
BACKGROUND
Blo t 11 is a high molecular weight allergen from Blomia tropicalis with significant immunoglobulin (Ig)E binding frequency. Native and recombinant Blo t 11 are susceptible to degradation and the isolation and expression of the allergen is problematic thus obtaining sufficient amounts of purified Blo t 11 for antibody production is limiting. DNA-based immunization is an attractive alternative strategy that bypasses antigen purification for antibody production.


OBJECTIVE
To use a DNA-based immunization protocol for the production and characterization of Blo t 11 monoclonal antibodies (mAbs).


METHODS
The 2625 bp cDNA coding for Blo t 11 was cloned into a mammalian expression vector and immunized intramuscularly with electroporation into mice. Monoclonal antibodies to Blo t 11 were generated using a methylcellulose-based hybridoma cloning kit. These mAbs were utilized for native Blo t 11 isolation and the development of sandwich enzyme-linked immunosorbent assay (ELISA).


RESULTS
Six mAbs recognizing the native and recombinant Blo t 11 were generated and characterized. Native Blo t 11 was affinity purified from Bt extract and its identity was confirmed by matrix assisted laser desorption/ionization - time of flight mass spectrometry. The native Blo t 11 showed IgE reactivity with 67% of mite allergic sera. A two-site ELISA developed showed a detection limit of 100 pg/ml of Blo t 11.


CONCLUSIONS
A DNA-based immunization protocol was successfully used to generate Blo t 11 mAbs with a spectrum of distinct epitopes located throughout the whole molecule, and they are useful for immunoaffinity purification and immunoassays.
Ramos
J D A
JD
Department of Paediatrics, National University of Singapore, Singapore.
Teo
A S M
AS
Lee
B W
BW
Cheong
N
N
Chua
K Y
KY
D000485:Allergens / Q000032:analysis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000917:Antibody Formation; D052179:Antigens, Plant; D004247:DNA / Q000276:immunology*; D004391:Dust; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes; D005260:Female; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007114:Immunization; D007073:Immunoglobulin E / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008925:Mites / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000276:immunology; D014335:Tropomyosin / Q000031:analogs & derivatives
D000485:Allergens; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; C448821:Blo t 11 allergen; D004391:Dust; D000939:Epitopes; D007106:Immune Sera; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E; D004247:DNA