Validation study of a new chemiluminescent singleplex IgE assay in a set of Italian allergic rhinitis patients

The measurement of specific IgE to allergenic extracts and molecules in patients with allergic rhinitis (AR) is crucial for a precise diagnosis and further immunotherapy. Companies providing in vitro diagnostic methods in allergology continuously strive for the optimization and modernization of such methods. A new generation of automated allergy tests based on chemiluminescence detection and paramagnetic microparticles is now available, with possible advantages in sample volume, cost‐effectiveness and avoidance of sample‐related interference.


| INTRODUC TI ON
Allergic rhinoconjunctivitis due to airborne allergens is the most prevalent immunological disease, with a particular impact among children and young adults. 1 Although it can be controlled with symptom-relieving drugs, specific allergy immunotherapy (AIT) is the only disease-modifying treatment with long-term effects currently available. Additionally, it allows for symptom control when pharmacotherapy fails. 2,3 The efficacy of AIT is based on the precise recognition of the allergen, as well as the allergenic molecules that elicit IgE sensitization and trigger patient's symptoms. [4][5][6] Although the prevailing diagnostic approach remains to be based on patients' clinical history combined with skin prick tests (SPT), 7 there are a plethora of situations where more precise information is required. In certain locations, like Southern European countries, it is often difficult to identify the eliciting allergen as patients are frequently multi-sensitized to multiple allergen sources with overlapping exposure, and often cross-reactive. 8 Component resolved diagnostics (CRD) using molecular level allergen components is a valuable tool that aids physicians in overcoming the diagnosis problem, as it allows one to identify the eliciting allergen and thus choose the most suitable agent for AIT. 5,9 Although considered too complex and detailed by many doctors, 10 the use of CRD to uncover the clinical relevance of IgE sensitization is mandatory when making a precise AIT prescription. In an allergic rhinitis clinical scenario, the doctor needs to establish a cause-effect relationship between exposure to the pollen recognized by the patient's IgE and the patient's symptoms 11 and precisely assess the degree of severity of the symptoms 12,13 since AIT is recommended for patients with moderate-severe rhinitis. 14 The ImmunoCAP® specific IgE assay, processed on Thermo Fisher (previously Phadia) equipment, has been widely adopted in Europe. It provides IgE tests against allergenic extracts or molecules (components) 15 and is often employed in the validation of new sIgE assays, 16 as well as comparison with other test systems. [17][18][19] In the ImmunoCAP sIgE assay, the allergen is covalently coupled during manufacturing to a flexible hydrophilic cellulose solid phase in a reaction vessel. 20 The NOVEOS System and specific IgE assay from HYCOR are novel in that they offer, among other advantages, a significantly lower (1/10th) sample volume per test in comparison with ImmunoCAP, and a robust chemistry design of chemiluminescence, fluorescence and paramagnetic microparticles which contribute to good precision and accuracy. 21 The assay design appears unaffected by known sample-related interferences including biotin, IgG, IgG4 and cross-reactivity by anti-carbohydrate determinants (CCD) antibodies that react with other cellulose-based technologies. [22][23][24] The aim of this study is to test, in comparison with ImmunoCAP, the analytical performance of the NOVEOS sIgE tests used in routine procedures.

| Study population
Consenting allergic rhinitis patients were consecutively recruited between 2016 and 2018 in the outpatient clinic of the Department of Pediatrics of "Sandro Pertini" Hospital in Rome and of the Allergy Unit, Istituto Dermopatico dell'Immacolata (IDI), Rome. All patients were tested for serum specific IgE antibodies against environmental allergens with routine tests (ImmunoCAP singleplex at Pertini Hospital and with ImmunoCAP ISAC at IDI). All IDI sera were retested with ImmunoCAP before inclusion in the present study. The present paper focuses on the comparison of the in vitro outcomes obtained by re-testing the study sera bank with the NOVEOS system and sIgE assay.

| Ethical approval
All participants and/or their parents or tutors gave their informed and written consent to the use of sera in scientific studies for the diagnosis and therapy of allergic diseases. The study design and procedures were approved by the local Ethical committees Comitato Etico Lazio 2 (#9871; 01/02/2016) for the Pertini Hospital and the Ethical Committee of IDI-IRCCS (#493/1; 30/05/2017).

K E Y W O R D S
allergic rhinitis, IgE, immunoassay, interassay comparison, precision medicine, singleplex Der p 2 (d203), D. farinae (d2), cat (e1), Fel d 1 (e94), dog (e5), horse (e3). Sera for comparison with the NOVEOS system were selected according to a 2:1 (pos:neg) ratio, on the basis of the ImmunoCAP ® outcome, for each allergen extract or molecule. The testing events and sera sets used for different allergens were therefore different.
In particular, 40 positive samples were selected from the available sera, with a randomization procedure targeted to obtain the whole range of sIgE levels between 0.35 kU/L and the highest value for that allergen. The negative sera were in contrast randomly selected, for each allergen, from the sera bank.

| NOVEOS IgE test
The NOVEOS test [ Figure 1] is a chemiluminescence detection system operating in a solid phase of fluorescently labelled and streptavidin-coated paramagnetic microparticles. The microparticles are first incubated with a biotinylated allergen that binds the streptavidin molecules. After an extensive wash, the bound microparticles are then incubated with patient serum containing allergenspecific IgE and the resulting bound complex is washed by aspirating unbound material from retained beads in the cuvette. They are subsequently incubated with an anti-IgE antibody conjugated to horseradish peroxidase and, after an incubation period, are washed to remove any unbound conjugate from bound material. The chemiluminescent signal is originated by adding a substrate solution. The concentration of allergen-specific IgE is directly proportional to the light intensity after correction (via fluorescence) for microparticle loss and is compared to an IgE reference curve traceable to World Health Organization (WHO) reference preparations (NIBSC 11/234).
The sample volume used per test is 4 µl, and the time to first result is 104 min.
F I G U R E 1 Fluorescently labelled and streptavidin-coated magnetic beads are incubated with a biotinylated allergen that bind to streptavidin on the surface of the beads. The allergen-coated beads are then incubated with patient serum containing allergen-specific IgE and, after an incubation period, are washed by aspirating unbound material from retained beads in the cuvette. The beads are subsequently incubated with an anti-IgE antibody conjugated to horseradish peroxidase and, after an incubation period, are washed to remove any unbound conjugate from bound material. The substrate solution is then added which generates a sustained chemiluminescence signal that is measured. The concentration of allergen-specific IgE is directly proportional to the light intensity after correction (via fluorescence) for any bead loss and is compared to an IgE reference curve traceable to World Health Organization (WHO) reference preparations [Colour figure can be viewed at wileyonlinelibrary.com]

| Statistical analysis
Age was summarized as mean and standard deviation (SD).
Categorical data were summarized as numbers (n) and frequencies (%). Sensitivity, specificity, positive and negative predictive values were calculated with their confidence interval at 95% (95% CI).
Accuracy, positive and negative likelihood ratios were also calcu-

| Study population and study design
Overall, 368 patients (208 in Pertini and 160 in IDI) (179 males; age 25 ± 15.2 years, ranging from 2 to 76 years of age) participated in this study (Table 1). Oral allergy syndrome (OAS) was the most frequent comorbidity, with a prevalence of 33.4% (123 patients), while asthma was diagnosed in 89 patients only (24.2%).

| Qualitative interassay comparison
Diagnostic overall performance criteria (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), accuracy, positive likelihood ratio (LR+), negative likelihood ratio (LR−)), that is, the outcomes of the NOVEOS sIgE compared to those of ImmunoCAP, were rather straightforward ( Table 2). NOVEOS pre-      Figure S1; Table 3).  that the NOVEOS test may be effectively used as an aid in in vitro diagnosis of IgE-mediated allergic diseases.

| Qualitative performance
The overall high sensitivity of NOVEOS sIgE tests in predicting a positive outcome of ImmunoCAP sIgE tests was accompanied by an even higher specificity across all the 21 reagents (15 extracts and 6 molecules). Molecules had higher sensitivity than extracts, while the inverse occurred for the specificity outcome, with molecules presenting a specificity of 94%. This was not surprising since the recombinant allergen molecules have a defined purity and biological activity. 26 In a preliminary study, 21

| Quantitative performance
The results of our qualitative analysis, based on a positive-negative

| Study limitations
We acknowledge a few limitations of our study. First, as the popula- Nevertheless, the allergens in this our study cover the vast majority of diagnostic needs of the European patient population affected by allergic rhinoconjunctivitis and asthma.

ACK N OWLED G EM ENTS
This study was supported by HYCOR (number 89772798), and HYCOR also provided the instrument and the reagents required to complete the study. Open access funding enabled and organized by ProjektDEAL.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.