[Highly sensitive enzyme-linked immunosorbent assay for the quantification of allergens from Dermatophagoides, DER p 1 and DER f 1].

AIM

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.

CONCLUSION

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.