The Establishment of EXiLE test for House Dust Mite Allergy

Abstract

Successful allergen immunotherapy (AIT) is associated with an increase in the production of allergen-specific IgG antibodies including IgG4 which may contribute to IgE-blocking factor (IgE-BF) activity. Recently, in vitro allergy test method was reported as EXiLE (IgE crosslinking-induced luciferase expression) test using a luciferase-reporting humanized rat mast cell line. However, to understand immunomodulation effect after AIT, this functional assay has not been fully validated. This study is aimed to investigate effect of IgE-BFs induced by house dust mite (HDM)-AIT on EXiLE test. HDM allergic rhinitis patients’ sera before- and after-AIT were used. EXiLE test was performed as previously reported (Allergol. Int, 2012). Briefly, RS-ATL8 cells were plated into the wells of a 96 well-plate and incubated with diluted serum overnight. The cells were stimulated with allergen (HDM, 0.1-1000 ng/mL) without washing. Then, luciferase substrate was added to the cells, and chemiluminescence was measured. The data was expressed as luciferase expression (LE) levels which were represented as the fold increase compared with the allergen untreated conditions (baseline), and EC50 value was calculated by fitting a dose response curve. The optimal incubation time was 3hr with HDM (0.1-1000 ng/mL). The LE levels showed significant correlation with the value of Der p specific-IgE (r2>0.95). EC50 of LE levels in sera after-AIT was shifted from that in sera before-AIT (p=0.01) EXiLE test was validated using the HDM allergic rhinitis sera. This test can be utilized for immunomodulation effect induced by AIT.