Detection of Multiple Allergen-specific IgEs on Microarrays by Immunoassay with Rolling Circle Amplification

Abstract

First described in 1967, the radio allergo sorbent test (RAST) has been the standard technique for measuring allergen-specific IgE antibodies in serum (1). An updated version of the RAST test, termed CAP (Pharmacia), has been introduced (2). In clinical practice, CAP results must be interpreted with care. The diagnostic performance of CAP varies in an allergen-specific manner, and CAP scores do not always correlate with clinical severity (3)(4). CAP sensitivity, specificity, and positive predictive values agree well with skin prick tests (SPTs) for house dust mites and grasses, but poorly with tests for cat dander and peanuts (5). Microarray technology potentially offers advantages in diagnostic applications such as allergy testing because the amount of reagent required, and thus the cost per assay, is greatly reduced (6). This approach has been difficult to reduce to practice, however, because the extremely small volumes (∼0.5–5 nL) of sample used to create spots on these microarrays require extremely sensitive methods of analyte detection (7). We have used rolling circle amplification (RCA) (8) for the detection of antibody bound to antigen (9). In this “immunoRCA”, the 5′ end of a RCA primer is attached to an antibody; thus, in the presence of circular DNA, DNA polymerase, and nucleotides, the rolling circle reaction produces a concatamer of circular DNA sequence copies that remain attached to the antibody. The amplified DNA can be detected by hybridization of complementary oligonucleotide probes. ImmunoRCA, therefore, represents a novel approach for signal amplification of antibody-antigen recognition events on microarrays. ImmunoRCA can detect IgE in a format using high-density microarrays of anti-human IgE printed on glass slides by a pin-tool type microarraying robot (9). Here, we describe the production of microarrays of multiple allergens and demonstrate the utility of these microarrays in combination with immunoRCA to simultaneously detect …