Molecular allergy diagnostics using multiplex assays: methodological and practical considerations for use in research and clinical routine

Abstract

The availability of single allergens and their use in microarray technology enables the simultaneous determination of specific IgE (sIgE) to a multitude of different allergens (> 100) in a multiplex procedure requiring only minute amounts of serum. This allows extensive individual sensitization profiles to be determined from a single analysis. Combined with a patient’s medical history, these profiles simplify identification of cross-reactivity; permit a more accurate estimation of the risk of severe reactions; and enable the indication for specific immunotherapy to be more precisely established, particularly in cases of polysensitization.Strictly speaking, a multiplex assay is not a single test, but instead more than 100 simultaneous tests. This places considerable demands on the production, quality assurance, and interpretation of data. The following chapter describes the multiplex test systems currently available and discusses their characteristics. Performance data are presented and the sIgE values obtained from multiplex and singleplex assays are compared. Finally, the advantages and limitations of molecular allergy diagnostics using multiplex assays in clinical routine are discussed, and innovative possibilities for clinical research are described.The multiplex diagnostic tests available for clinical routine have now become well established. The interpretation of test results is demanding, particularly since all individual results need to be checked for their plausibility and clinical relevance on the basis of previous history (patient history, clinical symptoms, challenge test results). There is still room for improvement in certain areas, for example with respect to the overall test sensitivity of the method, as well as the availability and quality of particular allergens. The current test systems are just the beginning of a continuous development that will influence and most likely change clinical allergology in the coming years.

EAACI Molecular Allergology User's Guide

“…From this initial proof of concept, the repertoire of allergens has increased, and the assays’ lower limit of quantitation and reproducibility have continued to improve. The commercially available version of this assay is the immune solid‐phase allergen chip or ISAC (ThermoFisher Scientific/Phadia) which requires 40 μl of serum to detect IgE antibody to 112 individual allergenic molecules that are in a static or planar array on a glass slide . The ISAC reports IgE antibody levels in ISU units, which are considered semiquantitative .…”

“…The commercially available version of this assay is the immune solid‐phase allergen chip or ISAC (ThermoFisher Scientific/Phadia) which requires 40 μl of serum to detect IgE antibody to 112 individual allergenic molecules that are in a static or planar array on a glass slide . The ISAC reports IgE antibody levels in ISU units, which are considered semiquantitative . A good correlation exists between the summed IgE anti‐cow's milk components levels (Bos d 4, 5, 6, and 8 and lactoferrin, r 2 = 0.66) as measured in 44 sera from clinically milk‐allergic individuals by the singleplex ImmunoCAP ( x ‐axis) and multiplex ISAC ( y ‐axis; Fig.…”

“…, right panel). The lower analytical sensitivity of the ISAC, however, depends on the allergen in question and is evident with many strongly positive IgE antibody levels as detected in the ImmunoCAP that are undetectable in the same sera when analyzed in the ISAC.…”

“…While the ISAC has been designed and thoroughly evaluated for use with purified recombinant and native allergenic molecules, other assays have employed allergen extracts immobilized in chip microarrays and multiplex assay modifications.…”

Performance evaluation of ImmunoCAP® ISAC 112: a multi-site study

“…The ImmunoCAP ® ISAC (Immuno Solid-phase Allergen Chip) sIgE 112 (ISAC 112) is a microarray-based immunoassay that enables simultaneous measurement of specific antibodies against an array of 112 recombinant or purified native allergen components derived from over 50 allergen sources [7][8][9]. Fluorescently labeled anti-IgE antibodies detect specific IgE from a serum or plasma test sample that binds to the immobilized allergen components.…”