Development of Monoclonal Antibody-Based EIA for Tetranor-PGDM which Reflects PGD2 Production in the Body.

Nanae Nagata,Tatsuro Nakamura,Takahisa Murata,Kosuke Aritake,Rikako Inoue,Sakura Masuko,Baohui Xu

doi:10.1155/2021/5591115

Nanae Nagata, Tatsuro Nakamura + Show 5 more

Open Access

https://doi.org/10.1155/2021/5591115

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Abstract

Tetranor-PGDM is a metabolite of PGD2. Urinary tetranor-PGDM levels were reported to be increased in some diseases, including food allergy, Duchenne muscular dystrophy, and aspirin-intolerant asthma. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength, pH, precision, and reliability. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited a half-maximal inhibition concentration (IC50) of 1.79 ng/mL, limit of detection (LOD) of 0.0498 ng/mL, and range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The intra- and inter-assay variation for tetranor-PGDM was 3.9–6.0% and 5.7–10.4%, respectively. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%–120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA. The established EIA would be useful for routine detection and monitoring of tetranor-PGDM in research or diagnostic body fluids.

Highlights

Tetranor-PGDM is a metabolite of PGD2 and reflects the biosynthesis of PGD2 in mice and humans [1]
As each urine sample from patients has different physicochemical features, we examined the optimal ion strength and pH for the MAbbased enzyme immunoassay (EIA)
IC50 values of 6.6, 5.7, 5.3, and 7.3 ng/mL were obtained with NaCl concentrations of 15, 75, 150, and 300 mM in the assay buffer, respectively

Summary

Introduction

Tetranor-PGDM is a metabolite of PGD2 and reflects the biosynthesis of PGD2 in mice and humans [1]. We previously reported that the urinary levels of tetranor-PGDM reflect the mast cell activity and severity of symptoms in patients with food allergies [2, 3]. Other studies reported that urinary tetranor-PGDM levels were elevated in other diseases, such as Duchenne muscular dystrophy [4] or aspirinintolerant asthma patients [5]. This urinary index improves the diagnosis and therapeutic procedure against these diseases. The measurement of urinary tetranor-PGDM requires liquid chromatography tandem mass spectrometry (LC-MS/MS). These instrumental analyses are expensive and require highly trained experts. The anti-tetranor-PGDM serumbased competitive enzyme immunoassay (EIA) kit is com-

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