title: A sensitive high throughput ELISA for human eosinophil peroxidase: a specific assay to quantify eosinophil degranulation from patient-derived sources.
abstract: Quantitative high throughput assays of eosinophil-mediated activities in fluid samples from patients in a clinical setting have been limited to ELISA assessments for the presence of the prominent granule ribonucleases, ECP and EDN. However, the demonstration that these ribonucleases are expressed by leukocytes other than eosinophils, as well as cells of non-hematopoietic origin, limits the usefulness of these assays. Two novel monoclonal antibodies recognizing eosinophil peroxidase (EPX) were used to develop an eosinophil-specific and sensitive sandwich ELISA. The sensitivity of this EPX-based ELISA was shown to be similar to that of the commercially available ELISA kits for ECP and EDN. More importantly, evidence is also presented confirming that among these granule protein detection options, EPX-based ELISA is the only eosinophil-specific assay. The utility of this high throughput assay to detect released EPX was shown in ex vivo degranulation studies with isolated human eosinophils. In addition, EPX-based ELISA was used to detect and quantify eosinophil degranulation in several in vivo patient settings, including bronchoalveolar lavage fluid obtained following segmental allergen challenge of subjects with allergic asthma, induced sputum derived from respiratory subjects following hypotonic saline inhalation, and nasal lavage of chronic rhinosinusitis patients. This unique EPX-based ELISA thus provides an eosinophil-specific assay that is sensitive, reproducible, and quantitative. In addition, this assay is adaptable to high throughput formats (e.g., automated assays utilizing microtiter plates) using the diverse patient fluid samples typically available in research and clinical settings.
authors: Division of Pulmonary Medicine, Department of Biochemistry and Molecular Biology, Mayo Clinic Arizona, Scottsdale, AZ, USA.
authors: Sergei I
authors: SI
authors: Ochkur
authors: John Dongil
authors: JD
authors: Kim
authors: Cheryl A
authors: CA
authors: Protheroe
authors: Dana
authors: D
authors: Colbert
authors: Rachel M
authors: RM
authors: Condjella
authors: Sophie
authors: S
authors: Bersoux
authors: Richard A
authors: RA
authors: Helmers
authors: Redwan
authors: R
authors: Moqbel
authors: Paige
authors: P
authors: Lacy
authors: Elizabeth A
authors: EA
authors: Kelly
authors: Nizar N
authors: NN
authors: Jarjour
authors: Robert
authors: R
authors: Kern
authors: Anju
authors: A
authors: Peters
authors: Robert P
authors: RP
authors: Schleimer
authors: Glenn T
authors: GT
authors: Furuta
authors: Parameswaran
authors: P
authors: Nair
authors: James J
authors: JJ
authors: Lee
authors: Nancy A
authors: NA
authors: Lee
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001249:Asthma / Q000175:diagnosis / Q000201:enzymology / Q000503:physiopathology; D001992:Bronchoalveolar Lavage Fluid / Q000737:chemistry; D015550:Cell Degranulation; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D047150:Eosinophil Cationic Protein / Q000378:metabolism; D047209:Eosinophil Peroxidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D047208:Eosinophil-Derived Neurotoxin / Q000378:metabolism; D004804:Eosinophils / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D017817:Nasal Lavage Fluid / Q000737:chemistry; D015203:Reproducibility of Results; D012220:Rhinitis / Q000175:diagnosis / Q000201:enzymology / Q000503:physiopathology; D012680:Sensitivity and Specificity; D012852:Sinusitis / Q000175:diagnosis / Q000201:enzymology / Q000503:physiopathology; D013183:Sputum / Q000201:enzymology
chemical_list: D000911:Antibodies, Monoclonal; D047209:Eosinophil Peroxidase; D047208:Eosinophil-Derived Neurotoxin; D047150:Eosinophil Cationic Protein