Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). Detection by enzyme-linked immunosorbent assay and purification from normal human urine.
Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) is one of the cationic proteins found in the granules of the human eosinophilic granulocytes. EPX was purified from extracts of granules isolated from blood buffy coat cells of healthy donors. Polyclonal anti-EPX antibodies were subsequently raised in rabbits. The anti-EPX-antibodies raised in rabbits showed no reactivity with other proteins in the granule extract. The sandwich ELISA utilized the biotin/avidin amplification system and measured EPX over the range of 60-2000 pg/ml. The intra- and interassay coefficients of variation were 6.5% and 8.2%, respectively, and the mean recoveries of 25 and 50 pg of purified EPX added to diluted serum samples were 106 +/- 16% (mean +/- SD; n = 12) and 112 +/- 14%, respectively. Using this assay we found high amounts of EPX in normal human urine (U-EPX). U-EPX was purified by a two step procedure involving affinity chromatography on heparin Sepharose and size exclusion chromatography on Sephadex G-50 superfine. Extracted EPX and U-EPX had ribonuclease activity and comigrated on agarose electrophoresis. They also showed immunological identity when evaluated with rabbit anti-EPX antibodies, but they differed slightly on SDS-PAGE probably due to differences in glycosylation. Our results support the findings that EPX/EDN is identical to a nonsecretory ribonuclease isolated from urine.
Reimert
C M
CM
Laboratory of Medical Allergology, University Hospital, Copenhagen, Denmark.
Minuva
U
U
Kharazmi
A
A
Bendtzen
K
K
D001798:Blood Proteins / Q000302:isolation & purification* / Q000652:urine; D001826:Body Fluids / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D047091:Eosinophil Granule Proteins; D047208:Eosinophil-Derived Neurotoxin; D004804:Eosinophils / Q000737:chemistry*; D006801:Humans; D007122:Immunoelectrophoresis; D009498:Neurotoxins / Q000302:isolation & purification* / Q000652:urine; D012260:Ribonucleases
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D009498:Neurotoxins; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases