Development of a Novel Enzyme-Linked Immunosorbent Assay for Blood and Urinary Eosinophil-Derived Neurotoxin: A Preliminary Study in Patients with Bronchial Asthma

Abstract

Background: Eosinophil-derived neurotoxin (EDN), also called eosinophil protein X (EPX), has been suggested to be a useful marker of eosinophilic inflammation. However, no commercial enzyme-linked immunosorbent assay (ELISA) kit for EDN is available yet. Methods: EDN was purified from pooled urine from healthy male volunteers. Polyclonal and monoclonal anti-EDN antibodies were subsequently raised, and a sandwich ELISA for EDN was established. EDN levels in serum, plasma and urine from asymptomatic patients with bronchial asthma were measured by the ELISA method. Some of the blood samples were also measured by a commercial radioimmunoassay (RIA) kit. Results: The ELISA method detected human EDN with a minimum detection limit of less than 0.62 ng/ml and did not cross-react with other eosinophil granule cationic proteins including eosinophil cationic protein. The intra- and interassay coefficients of variation of the ELISA method ranged from 2.6 to 3.6% and from 6.5 to 9.4%, respectively. Good linearity was observed with serially diluted different samples, and the recoveries of the purified EDN added to serum samples ranged from 85 to 110%. Median EDN concentrations in serum (36.9 vs. 19.1 ng/ml), plasma (23.0 vs. 14.5 ng/ml) and urine (118.2 vs. 72.1 μg/mmol Cr) were significantly raised in asymptomatic asthmatic patients compared with healthy control subjects. EDN levels in serum, plasma and urine from the patients significantly correlated with the number of peripheral blood eosinophils, but not total serum IgE levels. A significant relationship between EDN values measured by the EPX-RIA kit and the EDN-ELISA method was observed. Conclusions: We have developed a novel efficient ELISA method to specifically measure blood and urinary EDN, which may be useful to study the role of eosinophils in allergic diseases including bronchial asthma.