Quantitative measurement of serum allergen-specific IgE on protein chip

Abstract

Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease inflicting more than quarter of the world population. In order to identify allergen sources, skin provocation test and IgE serology was performed using allergen extracts. Such process identifies allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. Recently, microarray technology has been developed for allergen-specific IgE detection using rolling circle amplification. This study was carried out to evaluate protein chip technology for the quantitative measurement and limits of sensitivity of multiple allergen-specific IgE by an immunofluorescence assay. Significance of positive calibrators was tested using purified human IgE. Dermatophagoides pteronyssinus (Dp), egg white, milk, soybean, and wheat were used as allergens and human serum albumin as negative control. Sensitivity and clinical efficacy of protein chip were evaluated using allergy immune serum for Dp. The fluorescent intensities for purified human IgE as calibrator were well correlated with the concentrations of human IgE. Twofold dilution of serum allowed an optimal reaction with Dp (1 mg/ml) at which serum Dp-specific IgE levels by protein chip were compatible with those by UniCap®. The sensitivity of protein chip in this study was found at level of 1 IU/ml of IgE. Dp-specific IgE levels by protein chip correlated well with those of UniCap® by comparing 10 atopic dermatitis. Additional 18 sera were tested for above multiple antigens other than Dp and significant results were obtained for many antigens as well as Dp. These results indicated that spotting of heterogeneous protein mixture on protein chip and the quantitative measurement of serum allergen-specific IgE levels using immunofluorescence assay can be successfully applied in the clinical laboratory for the diagnosis of allergy and could be applied to diagnosis of autoimmune and infectious diseases

Optimization of Critical Factors Affecting the Performance of an Allergen Chip for the Analysis of an Allergen-specific Human IgE in Serum

“…3, IgE in serum was specifically bound to the respective allergen. A comparison of the results from the allergen chip with those from a CAP assay revealed a high correlation for two allergens (R 2 = 0.91 for two mites; 0.85 for D. farinae; 0.93 for D. pteronyssinus), which is comparable to a previous report, 2,6,8 as can be seen in Fig. 4A.…”

“…3,4,[6][7][8] Subsequently, the slide was washed with PBS, incubated with a solution of biotinylated anti-human IgE (Vector Laboratories, Burlingame, CA, USA) and 0.1% BSA in PBST (PBS containing 0.1% Tween-20) for 1 h, followed by a reaction with streptavidin-Cy3 conjugate (Sigma-Aldrich) for 1 h in the dark at room temperature. The slide was then washed three times with PBST and distilled water, and dried with N2 gas before scanning.…”

“…When considering the increasing number of allergens and very low abundance of IgE in serum (10 -3 -10 -4 fold of immunoglobulin G), 5 the development of a more multiplexed and sensitive assay method is highly required. 6 Recently, chip-based analyses have been attempted in conjunction with various detection methods, such as rolling circle amplification, 7 chemiluminescence, 4 and fluorescence 6,8,9 as a promising approach, because it enables a sensitive and high-throughput assay with tiny amounts of reagents. 10 As capturing agents of the chip-based analysis of an allergy, allergen extracts 6 or purified allergen molecules 8 were of preferred use.…”

“…6 Recently, chip-based analyses have been attempted in conjunction with various detection methods, such as rolling circle amplification, 7 chemiluminescence, 4 and fluorescence 6,8,9 as a promising approach, because it enables a sensitive and high-throughput assay with tiny amounts of reagents. 10 As capturing agents of the chip-based analysis of an allergy, allergen extracts 6 or purified allergen molecules 8 were of preferred use. Of them, purified allergens produced by recombinant DNA techniques often resulted in incorrect folding or missing glycosylation required for IgE recognition, 11,12 which causes a loss of some IgE-binding epitopes.…”

Human body fluid proteome analysis

“…An allergen microarray containing 94 purified allergen molecules that represent the most common allergen sources was developed for monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens using only minute amounts of serum [126]. This microarray test provides equivalent performance to ELISA tests and offers a significant advantage in convenience and cost when compared to traditional allergy test formats [126][127][128][129][130][131][132]. Protein arrays using Hepatitis B or C virus (HBV and HCV) antigens were also described for detection of HBV or HCV antibodies in patients' sera [133,134].…”