4. Advantages and Limitations of IgE Antibody Microarray Assays.
Figure 1 depicts a number of possible singleplex and microarray assay configurations that can be employed with recombinant and natural allergen molecules. The most attractive microarray configuration involves spotting purified allergens individually on activated glass chips for multiple specificity IgE antibody screening. Most recently, the current ISAC assay has 112 individual allergenic molecules spotted in triplicate. A research version of this assay called the Mechanisms of the Development of ALLergy (MeDALL) Allergen Chip expanded the number of allergenic molecules immobilized on the chip to 170 [16]. It was used to study IgE antibody sensitization profiles of European birth cohorts. Certainly, the principal advantage of the multiplex microarray chip is its impressive technology that permits the detection of IgE antibody to a broad spectrum of clinically relevant allergenic molecules using a small quantity of serum. As suggested above, there are limitations to the microarray-based panel testing format. It encourages abuse by performing measurements of unwanted or unneeded IgE antibody specificities that are not indicated by the patient’s clinical history. Moreover, the chip-based microarray is less quantitative and potentially less analytically sensitive than the singleplex autoanalyzers that are in widespread use throughout the world. Chip-based microarrays are more difficult to quality control due to the number of different allergens on a single solid phase surface. They pose an increased risk for greater inter-lot variability due to the need to perform simultaneous quality control on over 100 different allergen molecules (or extracts). Possibly of most concern, chip microarrays are subject to interference by allergen-specific IgG antibodies due to the low level of allergen deposited on the chip per spot. This can compromise the quantitative accuracy and bias the analysis toward only high affinity IgE antibody. Alternatively, the interference can be viewed positively as a surrogate marker for the result of specific immunotherapy that elicits IgG-blocking antibody [17].
The advantage of the IgE antibody chip-based microarray rests in its conservation of serum volume, increased speed of analysis, reduced technician intervention and the use as an optimal configuration for point-of-care tests. The availability of recombinant allergenic molecules has enhanced IgE antibody assay performance in a number of ways. First, it has permitted an increased analytical sensitivity by supplementing extracts that are deficient in a particular allergen specificity (e.g., Cor a 1 for hazelnut; Hev b 5 for Hevea brasiliensis natural rubber latex). Second, there is an increase in analytical specificity when selected molecules are used to detect IgE antibodies. For instance, Ara h 2 for peanut; Cor a 9 and 14 for hazelnut detect IgE antibody profiles that aid in determining a clinical risk for a systemic reaction and clarifying the need for an oral food challenge. Third, molecular allergens allow the identification of cross-reactivity (e.g., pan allergens Phl p 7 [polcalcin] and Phl p 12 (profilin) in Timothy grass, lipid transfer proteins in plant food and pollens [18]). And fourth, selected molecules define IgE antibodies as a marker of a genuine primary sensitization. (e.g., Fel d 1 for cat; Ves v 1 and Ves v 5 for yellow jacket venom). The microarray format allows simultaneous IgE antibody testing of multiple structurally similar allergens and thus allows more time and cost-effective evaluation of possible IgE antibody binding to the principal families of cross-reactive allergen molecules (e.g., Pathogenesis-Related proteins (PR10 family), tropomyosins, serum albumins, non-specific lipid transfer proteins, profilins, polcalcins, and seed storage proteins).