Detection of Allergen-specific IgE on Microarrays by Use of Signal Amplification Techniques

Abstract

Allergy is estimated to be the sixth leading cause of chronic disease in the US, and the number of people exhibiting symptoms of an allergic response to various natural and synthetic compounds has increased dramatically in the last decade (1). The prevalence of food allergies is particularly evident in children; some reports estimate that the frequency of food allergies in infants is 2–5% (2). The identification of the specific IgE responsible for the clinical symptoms can be a costly and lengthy procedure. Various immunoassays, such as ELISA (3), the Radio Allergo Sorbent Test (4), and high-capacity solid-phase tests, e.g., the CAP system (Pharmacia) (5), are currently used in the diagnosis of allergies and have the inherent sensitivity and specificity to detect IgE in human serum. However, all of these assays are time-consuming, require large quantities of serum samples, and use poorly characterized antigen preparations. Moreover, none of the assays currently used in the diagnosis of allergies has the throughput to screen for the most common allergens, which are estimated to exceed 300. High-density ordered arrays of molecules (microarrays) (6)(7) may circumvent most of the current limitations in the diagnosis of allergies by allowing simultaneous and multiparametric analysis of serum reactivity against a variety of antigens. Protein microarrays in combination with fluorochrome-labeled secondary antibodies have been used to reveal the presence in human serum of IgG and IgM directed against microbial antigens (8). However, this assay format is not suitable for the serodiagnosis of allergies. A signal enhancement procedure is required to reveal the presence of subnanomolar concentrations of analytes, such as serum-specific IgE. One such method has recently been described (9); here we report on an alternative methodology. We developed a high-sensitivity assay to reveal the presence of specific IgE in human sera that combines allergen microarrays with …