Page 1329 - Clinical Immunology_ Principles and Practice ( PDFDrive )

1290 Part ElEvEn Diagnostic Immunology

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Gly m 4-soybean, Ara h 8-peanut, Aln g 1-alder, Act d 8-kiwi, immobilized in triplicate microdot arrays. The present day
Api g 1-celery, and Dau c 1-carrot. Other component based version of this assay is the immune solid phase allergen chip or
cross-reactivity groups include the profilins, nonspecific lipid ISAC (Thermo Fisher Scientific/Phadia, Uppsala, Sweden) in
transfer proteins tropomyosins, serum albumins, polcalcins, which 40 µL of serum is applied to a chip that has 112 individual
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lipocalins, parvalbumins, storage-binding proteins, and carbo- allergenic molecules immobilized. IgE anti–cow’s milk com-
hydrate cross-reactive determinants (CCDs) (see Table 95.2). ponents as measured in the multiplex ISAC have agreed well
Each of these cross-reactive allergen families is extensively dis- with those obtained in a single-plex autoanalyzer using the
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cussed in the Handbook on Molecular Allergology. 44 same allergen specificities. Other groups have tried alternative
multiplexing technologies to detect IgE antibodies that have
Calibration included immobilizing allergen extracts on chips by using
The second attribute that varies widely among commercially Luminex bead–based suspension arrays, employing nanotechnol-
available IgE antibody assays is calibration algorithm and ogy biosensors, detecting surface plasmon resonance, and using
methodology. There exists no internationally recognized poly- plates equipped to produce electrical pulse generated chemilu-
clonal human IgE antibody reference preparation. Thus all three minescence. 51,52 When selecting a single-plex or multiplex assay
autoanalyzers currently in commercial use in the United States format, one must consider their strengths and weaknesses.
have found a heterologous interpolation procedure to be optimal Single-plex systems have the advantage of greater analytical
in which allergen-specific IgE antibody response data are inter- sensitivity or a lower limit of quantitation, greater precision and
polated from a total serum IgE calibration curve. This procedure accuracy, more established internal and external quality control,
is valid as long as the total IgE calibrators and the patient’s and wider global availability of technology. In contrast, multiplex
allergen-specific IgE antibody levels dilute out in parallel so that systems provide increased speed of analysis with reduced turn-
parallelism is maintained. The assays report IgE antibody levels around times, conservation of sample volume, greater simplicity,
in the same units (kUa/L), using internal total IgE calibrators and reduced technical and reagent costs. 52
that are cross-verified and traceable to the World Health Organiza- The quality of allergen-specific IgE antibody results reported
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tion (WHO) 11/234 IgE international reference preparation. from clinical diagnostic allergy laboratories is not uniformly
This calibration system allows interpolation of IgE antibody equivalent. In addition to the variability of results for a given
results from a limit of quantitation of 0.1 kUa/L to 100 kUa/L serum between assays from different manufacturers as a result
levels of IgE antibody. In terms of quantitation, at least one of of allergen and calibration variance, the positive thresholds used
the IgE antibody autoanalyzers (ImmunoCAP) has demonstrated for the same assay by different laboratories varies (e.g., 0.1, 0.35,
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equivalence in which 1 kUa/L of chimeric allergen-specific IgE and 0.7 kUa/L). For this reason, physicians requesting IgE
antibody was shown to be equivalent to 1 kU/L (2.4 ng/mL) of antibody testing bear some responsibility for determining the
total serum IgE. 46 quality of the results they receive. In the United States, testing
should be performed in a clinical laboratory that is federally
Single-Plex, Multiallergen, and Multiplex Assays licensed for highly complex immunology clinical testing under
The autoanalyzers in common clinical use are single-plex (or CLIA-88 (verified by requesting a copy of the federal laboratory
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monoplex) assays in which one analyte (e.g., IgE anti–cat dander) license). The requesting physician should inquire about the
is measured in a single analysis. In contrast, a multiplex antibody assay method used, the source of its reagents, and how assays
assay allows many specificities of a single antibody isotype (e.g., are quality controlled by the laboratory. As part of the formal
IgE) to be individually detected and semiquantified in a single record, the assay method used in patient analysis should be
analysis. These are distinguished from a multiallergen screening indicated on the final report.
assay, which is a form of single-plex analysis that involves the
use of a mix of multiple allergens that is immobilized on the Competitive IgE Antibody Inhibition Assay
same allergosorbent. The purpose of the multiallergen assay is The competitive inhibition format of the IgE antibody assay has
to simultaneously screen a serum for IgE antibody to a concise been used by researchers, allergen manufacturers, and regulators
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number (e.g., 10–15) allergens either of the same allergen source to determine the relative potency of allergen extracts. One
type (e.g., foods: chicken egg, cow’s milk, peanut, soybean, cod practical research application of the IgE antibody inhibition assay
fish) or diverse sources (e.g., respiratory allergens as an aeroal- has been as a tool for monitoring the concentration of allergens
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lergen mix: pollen from select trees, grasses, weeds, pet epidermals, released into environments (e.g., latex allergen in hospital air ;
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dust mites, molds). A qualitative result (positive or negative) air sampling of airplanes for aerosolized peanut allergen). In the
is generated, and it serves as an inexpensive and efficient single diagnostic allergy laboratory, the competitive IgE antibody
analysis to assess the general atopic status (IgE positivity) of an inhibition assay has been used analytically to confirm an IgE
individual. One multiaeroallergen screening assay is being used antibody assay’s minimum detectable dose (sensitivity) and
to assess the atopic status (genetic predisposition to produce IgE nonspecific binding and to document the extent of cross-reactivity
antibody) of research subjects enrolling in clinical studies of by determining the allergen specificity of IgE antibody. 13
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asthma. Clinically, the multiaeroallergen screening assay has a The one clinical application of the IgE antibody inhibition
high negative predictive power and thus is used to rule out assay has been as an adjunct to define the appropriate therapeutic
IgE-mediated allergic disease where the suspicion based on the composition of venoms for patients who have insect-sting allergy
clinical history is weak. with multiple potentially cross-reactive sensitivities and who
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The availability of unlimited quantities of molecular allergens have elected to receive immunotherapy. Indications for this
has made it possible to develop multiplex chip based microarrays test include a strong skin reactivity; a high level of serum IgE
for diagnostic allergy confirmatory testing. The first microarray antibody to yellow jacket venom (YJV) and a weak skin reactivity;
for semiquantification of IgE antibody involved a preactivated or a low level of serum IgE antibody specific for Polistes wasp
glass slide or chip on which 94 purified allergens were each venom (PWV). The structural similarity between Vespid and