Summary
An enzyme immunoassay (EIA) capable of measuring urinary tetranor‐PGDM concentrations was developed.
The EIA distinguished patients with food allergy from healthy individuals based on urinary tetranor‐PGDM concentrations.
Keywords: enzyme immunoassay, food allergy, tetranor‐PGDM
To the Editor,
The prevalence of food allergies has been increasing, particularly among children. During food allergic reactions, mast cells release prostaglandin D2 (PGD2), whose urinary metabolite, 9α‐hydroxy‐11,15‐dioxo‐13,14‐dihydro‐2,3,4,5‐tetranor‐prostan‐1,20‐dioic acid (tetranor‐PGDM) [1], serves as an objective and specific indicator of allergic symptoms during oral food challenges (OFCs) [2, 3, 4] and desensitisation in oral immunotherapy [5]. Tetranor‐PGDM is excreted in urine, which can be collected non‐invasively. To enable versatile measurement of tetranor‐PGDM, we developed a monoclonal antibody–based enzyme immunoassay (EIA) [6]. This study aimed to evaluate the utility of this EIA for measuring urinary tetranor‐PGDM concentrations and to assess whether these values could distinguish patients with food allergy from healthy individuals.
The antibody used in the assay exhibits higher affinity for 8‐((1R,2S)‐2‐(2‐carboxyethyl)‐5‐oxocyclopent‐3‐en‐1‐yl)‐6‐oxooctanoic acid (tetranor‐PGJM), with markedly lower cross‐reactivity for other PGD2 metabolites than for tetranor‐PGDM. Additional results are shown in online repository material (https://zenodo.org/records/18615641). Tetranor‐PGDM is converted into tetranor‐PGJM through dehydration. The calibration curve was constructed using tetranor‐PGJM concentrations ranging from 0.02 to 5 ng/mL, providing sufficient sensitivity for detecting urinary tetranor‐PGJM levels. Interday (five replicates) and intraday (5 days) variations were assessed at concentrations of 0.32, 0.8 and 2 ng/mL, corresponding to the steepest part of the calibration curve. The coefficient of variations (CVs) for interday and intraday experiments ranged from 2.2% to 3.2% and 1.9% to 4.5%, respectively. Recovery rates, evaluated using tetranor‐PGJM–spiked phosphate‐buffered saline, ranged from 95.7% to 105.4%, confirming the assay's reliability.
Urine samples were collected following protocols approved by the ethics committees of the National Center for Child Health and Development (2017, No. 2127) and the University of Tokyo (1 May 2014, No. 14‐25). All participants provided informed consent. A total of 42 participants aged 4–14 years were enrolled, including 28 healthy children (without food allergies, bronchial asthma, atopic dermatitis or hay fever) and 14 children with food allergy scheduled to undergo OFCs at the Division of Allergy, National Center for Child Health and Development. Urine samples were collected from patients 4 h after OFC, and from healthy individuals before taking a regular meal. Patients exhibiting acute allergic symptoms beyond oral symptoms during OFCs were classified as OFC‐positive. Some OFC‐positive patients also had other allergic conditions (bronchial asthma: 28.6%; atopic dermatitis: 71.4%; allergic rhinitis: 85.7%). The administered allergens included egg white (50%), nuts (43%) and wheat (7%). Allergic reactions were scored on a scale of 1–5, according to criteria published by Sampson [7]. Seven patients (50% of the OFC‐positive patients) scored 2, and the other seven patients (50%) scored 3. There were no patients who scored 1, 4 or 5.
Urine samples were centrifuged (20,400 × G, 5 min, 4°C), diluted (1:10) with 150 mM of sodium phosphate buffer (pH 9.0) and heat‐treated (100°C, 30 min) to convert tetranor‐PGDM into tetranor‐PGJM. The samples were then purified by methanol and solid‐phase extraction. The conversion efficiency of tetranor‐PGDM–spiked urine quantified by liquid chromatography‐tandem mass spectrometry (LC–MS/MS) was 100.2% ± 4.3% with minimal intersample variability (CV: 9.7%). Urinary tetranor‐PGJM concentration was measured using the EIA. For the EIA, 60 ng/mL of tetranor‐PGJM (Cayman Chemical, MI, USA) was coated onto an amino group‐coated 96‐well EIA plate (Sumitomo Bakelite, Tokyo, Japan). After blocking the wells, purified urine samples and 50 ng/mL of horseradish peroxidase–conjugated anti‐tetranor‐PGJM antibody (developed in‐house) were added to the wells.
The urinary tetranor‐PGJM concentrations measured by the EIA showed a positive correlation with values measured by LC–MS/MS (Pearson r = 0.82, p < 0.001 performed with R software), confirming the clinical applicability of the EIA. In the healthy group, urinary tetranor‐PGJM concentrations ranged from 0.00 to 5.31 ng/mg creatinine, with a mean of 2.53 ng/mg creatinine (Figure 1A). In the OFC‐positive group, concentrations ranged from 1.69 to 27.26 ng/mg creatinine, with a mean of 10.30 ng/mg creatinine. The OFC‐positive group showed significantly higher values than the healthy group (p < 0.001, Mann–Whitney test performed with R software). To evaluate the diagnostic potential of EIA for food allergy, a receiver operating characteristic (ROC) curve analysis was performed (Figure 1B). The area under the curve was 0.91. The cut‐off value for distinguishing OFC‐positive patients from healthy individuals was 4.07 ng/mg creatinine. Sensitivity and specificity were 0.93 and 0.82, respectively.
FIGURE 1.
Urinary tetranor‐PGJM concentration measured by EIA. (A) Relationship of urinary tetranor‐PGJM concentrations measured by EIA between healthy individuals (n = 28) and OFC‐positive food allergy patients (n = 14). ***p < 0.001, Mann–Whitney U test. (B) Receiver operating characteristic curve for urinary tetranor‐PGJM concentrations with healthy individuals versus OFC‐positive food allergy patients. AUC, area under the curve.
In this study, EIA values for the OFC‐positive group were significantly higher than those for the healthy group, with a cut‐off value of 4.07 ng/mg creatinine for differentiation. We focused on children aged 4–14 years, as they were able to provide urine samples independently. The cut‐off value obtained in this study was higher than those reported in previous studies using LC–MS/MS [2]. This discrepancy may reflect the binding of degradants with preserved epitope derived from tetranor‐PGDM and/or tetranor‐PGJM to the antibody used in the EIA.
In summary, this study demonstrates the feasibility of using an EIA to predict food allergic reactions based on urinary PGD2 metabolite levels. These findings provide a foundation for the objective evaluation of food allergic reactions.
Author Contributions
T.M., K.Y.‐H., Y.O. and N.N. designed the experiments and supervised the project. T.I., S.M., R.I. and M.F. performed the experiments and analysed the data. T.N., K.A. and S.O. provided expertise in the study methodology. H.O., M.S., Y.I., K.U., S.H., T.F., Y.O. and K.Y.‐H. provided patients' samples and clinical information. T.I., N.N. and T.M. wrote the manuscript. All authors provided feedback on the manuscript.
Funding
This work was supported by Grant‐in‐Aid for Scientific Research from Japan Society for the Promotion of Science [20H05678 and 25H00430 to T.M., 22K05984 to N.N.]; an Adaptable and Seamless Technology Transfer Program through Target‐driven R&D (A‐STEP) from the Japan Science and Technology Agency [JST, JPMJTR22UF, to T.M.]; and Nipponham Foundation [to K.Y.‐H.].
Conflicts of Interest
T.M. belongs to an endowed course (Food and Animal Systemics) provided by the Revamp Corporation (Tokyo, Japan). All other authors have no conflicts of interest.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.