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.

A prospective analysis evaluating tissue biopsy location and its clinical relevance in chronic rhinosinusitis with nasal polyps

“…ECP is an eosinophil granule protein that correlates well with cytokines (eg, interleukin 5 [IL-5] and IL-13) and chemokines (eg, eotaxin-2/CCL24 and eotaxin-3/CCL26) as well as blood and tissue eosinophilia. 9, 24, 25 In prior studies, we found that NP has higher levels of ECP than uncinate tissue (UT) even among patients with CRSwNP. More recently, we found that although ECP levels measured in NP were higher than in UT, UT ECP levels were moderately to strongly correlated with radiographic severity and other type-2 mediators.…”

Rapid quantification of sputum eosinophil peroxidase on a lateral flow test strip

“…[4][5][6] A paper-based colorimetric LFT was developed using the wellknown antibody sandwich format ( Figure 1A). 5 As expected, samples from patients with "controlled" inflammation, that is, sputum eosinophils < 3% of total cells (with a total cell count less than 9.7 million cells) 8 showed lower EPX content compared to the "eosinophilic" group with > 3% eosinophils ( Figure 1D, P = .028). The LFTs were imaged using a Flip-Pal (#100C) handheld scanner 15 min after sample addition (details of method development and materials have been provided in the online repository).…”

Development of a suspension array assay in multiplex for the simultaneous measurement of serum levels of four eosinophil granule proteins

“…Although levels of MBP, ECP, EDN and EPO in serum, other body fluids, and cell supernatants can be measured by radioimmunoassay (RIA) (Wassom et al, 1981; Peterson et al, 1991; Robinson et al, 1995) and enzyme-linked immunosorbent assay (ELISA) (Reimert et al, 1991b; Yamamoto et al, 1994; Morioka et al, 2000; Ochkur et al, 2012), both of these methods are time-consuming and measure each granule protein individually. Suspension array technology has the potential to circumvent both of these problems and has been used for the sensitive and simultaneous measurement of multiple proteins in a wide variety of clinical samples, including serum, plasma, cerebrospinal fluid (Moller, 2012), sputum (Bafadhel et al, 2012) and urine (Campbell et al, 2013).…”