[Highly sensitive enzyme-linked immunosorbent assay for the quantification of allergens from Dermatophagoides, DER p 1 and DER f 1].
OBJECTIVE
In order to specifically quantify the two major Dermatophagoides spp. allergens, Der p 1 and Der f 1, separately, we tried to establish a highly sensitive enzyme-linked immunosorbent assay (ELISA).


METHODS
Ninety-six-well ELISA plates were coated with mouse monoclonal antibodies specific against Der p 1 or Der f 1. Allergen samples were incubated in the wells for 2 hours at 37 degrees C. After washing with PBS-T, biotinylated rabbit anti-Der 1 polyclonal antibody was added to the wells. The allergens were detected using horse radish peroxidase-conjugated streptavidin, an enzyme substrate (TMB/H2O2) and a microplate reader.


RESULTS
The working range of both ELISA systems for Der p 1 and Der f 1 was 40-2500 pg/ml. The intra- and inter-assay coefficients of variation for reproducibility were 0.99-4.38% and 0.68-3.02%, respectively, in Der p 1 ELISA and 1.54-3.65% and 0.39-4.77%, respectively, in Der f 1 ELISA. Moreover, these ELISA systems showed that there was no cross-reactivity between Der p 1 and Der f 1 allergens.


CONCLUSIONS
These ELISA systems may be useful for measuring less than 1 ng/ml of major mite allergens in house dust samples, various pharmaceutical studies such as evaluation of an allergen-inactivating agent, and standardizing recombinant/natural Dermatophagoides spp. allergens.
Kondoh
Masatoshi
M
Central Research Laboratories, Nichinichi Pharmaceutical Corporation Ltd. lab@nichinichi-phar.co.jp
Fukada
Kazutake
K
Shimada
Takashi
T
Kitamura
Yoshihisa
Y
Yasueda
Hiroshi
H
Enomoto
Tadao
T
D000485:Allergens / Q000032:analysis*; D039741:Antigens, Dermatophagoides / Q000032:analysis*; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*
D000485:Allergens; D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; D003546:Cysteine Endopeptidases; C451159:Dermatophagoides farinae antigen f 1; C451162:Dermatophagoides pteronyssinus antigen p 1