Enhanced sensitivity of capture IgE-ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens

The detection of allergen-specific immunoglobulin (Ig)E is an important method for the diagnosis of IgE-mediated allergic diseases. The sensitivity of the indirect IgE-ELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts. To overcome these limitations, a novel capture IgE-ELISA based on a recombinant Der f 1/Der f 2 fusion protein (rDer f 1/2) was developed to enhance the sensitivity to IgEs that bind allergens from the house dust mite (HDM) species Dermatophagoides farina. pET28-Der f 1/2 was constructed and expressed in Escherichia coli BL21 (DE3) pLysS. The purified fusion protein was evaluated by IgE western blotting, IgE dot blotting and indirect IgE-ELISA. Capture-ELISA was performed by coating wells with omalizumab and incubating in series with sera, biotinylated Der f 1/2, horseradish peroxidase-conjugated streptavidin and 3,3,5,5-tetramethylbenzidine. The relative sensitivities of indirect-ELISA and capture-ELISA for HDM allergen-specific IgE binding were determined; sera from non-allergic individuals were used as the control group. rDer f 1/2 was expressed in the form of inclusion bodies comprising refolded protein, which were then purified. It exhibited increased IgE-specific binding (24/28, 85.8%) than rDer f 1 (21/28, 75.0%) or rDer f 2 (22/28, 78.6%) with HDM-allergic sera. Furthermore, in a random sample of HDM-allergic sera (n=71), capture-ELISA (71/71, 100%) was more sensitive than indirect-ELISA (68/71, 95.8%) for the detection of HDM-specific IgEs (P<0.01), indicating that this novel method may be useful for the diagnosis of HDM allergy.