Endogenous peroxidases in sputum interfere with horse-radish peroxidase-based ELISAs

Kim, Lisa Ha Yeon; Plaza, Karin Lazo; Thomas, Sruthi; Draijer, Christina; Radford, Katherine; Peters‐Golden, Marc; Mukherjee, Manali; Nair, Parameswaran

doi:10.1016/j.jim.2017.11.005

Cited by 8 publications

(6 citation statements)

References 18 publications

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“…Lower limits of detection were 9.38 pg/mL and 3.12 pg/mL, respectively. Both ELISAs were performed following the user’s manuals with substitution of Strepavidin alkaline phosphatase rather than the Strepavidin horseradish peroxidase provided in the kits [ 20 ].…”

Section: Methodsmentioning

confidence: 99%

Iron in airway macrophages and infective exacerbations of chronic obstructive pulmonary disease

Nichols

Nair

et al. 2022

Respir Res

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Background Excess pulmonary iron has been implicated in the pathogenesis of lung disease, including asthma and COPD. An association between higher iron content in sputum macrophages and infective exacerbations of COPD has previously been demonstrated. Objectives To assess the mechanisms of pulmonary macrophage iron sequestration, test the effect of macrophage iron-loading on cellular immune function, and prospectively determine if sputum hemosiderin index can predict infectious exacerbations of COPD. Methods Intra- and extracellular iron was measured in cell-line-derived and in freshly isolated sputum macrophages under various experimental conditions including treatment with exogenous IL-6 and hepcidin. Bacterial uptake and killing were compared in the presence or absence of iron-loading. A prospective cohort of COPD patients with defined sputum hemosiderin indices were monitored to determine the annual rate of severe infectious exacerbations. Results Gene expression studies suggest that airway macrophages have the requisite apparatus of the hepcidin-ferroportin axis. IL-6 and hepcidin play roles in pulmonary iron sequestration, though IL-6 appears to exert its effect via a hepcidin-independent mechanism. Iron-loaded macrophages had reduced uptake of COPD-relevant organisms and were associated with higher growth rates. Infectious exacerbations were predicted by sputum hemosiderin index (β = 0.035, p = 0.035). Conclusions We demonstrate in-vitro and population-level evidence that excess iron in pulmonary macrophages may contribute to recurrent airway infection in COPD. Specifically, IL-6-dependent iron sequestration by sputum macrophages may result in immune cell dysfunction and ultimately lead to increased frequency of infective exacerbation.

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Section: Methodsmentioning

confidence: 99%

Iron in airway macrophages and infective exacerbations of chronic obstructive pulmonary disease

Nichols

Nair

et al. 2022

Respir Res

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“…And then H 2 O 2 is used in the second enzymatic reaction by peroxidase to form a chromogen which is quantitatively measured at a wavelength of 515 (or 520) nm. Although this enzymatic assay is simple and commercially available, the chromogen formation by peroxidase-catalyzed reaction of H 2 O 2 is susceptible to interference from endogenous confounding components in biological matrixes, and eventually may cause false positive or negative results [19][20][21][22] . The Salimetrics® assay requires centrifugation to remove mucins and other interferents from saliva samples, but it cannot perfectly eliminate multiple compounds that can act as interferers.…”

Section: Salivary Ua Levels In Patients With Gout Compared With Non-g...mentioning

confidence: 99%

“…However, they are not suitable for daily personal use because they are usually time-consuming and requiring expensive instruments and experts. Although some commercially available enzymatic assay kits can be used for various types of biological matrices such saliva, serum, and urine 2 , they can be influenced by other interferences such as vitamin C, lipid, or endogenous peroxidase that are present in biological samples, and eventually cause false results 2,[19][20][21][22] . Besides, they have a short use-by date because peroxidase within reagents is incompatible with preservatives such as sodium azide 23 .…”

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confidence: 99%

Electrochemical detection of uric acid in undiluted human saliva using uricase paper integrated electrodes

Han

Kang

et al. 2022

Sci Rep

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In this study, we introduce a uricase-immobilized paper (UOx-paper) integrated electrochemical sensor for detection of uric acid (UA) in saliva. The UOx was immobilized on the detection zone in the wax-patterned paper substrate. This UOx-paper was integrated with a Prussian blue-modified, screen-printed carbon electrode after electropolymerization of o-phenylenediamine to construct an electrochemical cell for small-volume (20 μL) of samples. First, we optimized the fabrication conditions of UOx-paper. Next, the amperometric response of the UOx-paper-based electrochemical UA sensor was analyzed using a known concentration of UA standard solution in artificial saliva at an applied potential of − 0.1 V (versus Ag pseudo-reference electrode). The UOx-paper based electrochemical UA sensor showed a sensitivity of 4.9 μA·mM−1 in a linear range of 50 to 1000 μM (R2 = 0.998), high selectivity and good reproducibility, as well as a limit of detection of 18.7 μM (0.31 mg/dL) UA. Finally, we quantified the UA levels in undiluted saliva samples of healthy controls (n = 20) and gout patients (n = 8). The levels were correlated with those measured with conventional salivary UA enzymatic assays as well as serum UA levels. The UOx-paper-based electrochemical UA sensor is a user-friendly and convenient tool to assess salivary UA levels.

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“… 52 However, we need to exercise caution with the interpretation of the human data, since the sputum ANAs reported were detected using a horse radish peroxidase-based ELISA platform, which might not be ideally suited for sputum samples. 53 Nevertheless, the possible presence of luminal ANAs in COPD patients' refractory to steroid treatment further highlights the importance of identifying tissue-specific autoimmune mechanisms latent in obstructed airways.…”

Section: Arriving At a Hypothesis For Autoimmune Pathomechanism In Asmentioning

confidence: 99%

Autoimmune Responses in Severe Asthma

Mukherjee

Nair

2018

Allergy Asthma Immunol Res

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Asthma and autoimmune diseases both result from a dysregulated immune system, and have been conventionally considered to have mutually exclusive pathogenesis. Autoimmunity is believed to be an exaggerated Th1 response, while asthma with a Th2 underpinning is congruent with the well-accepted Th1/Th2 paradigm. The hypothesis of autoimmune involvement in asthma has received much recent interest, particularly in the adult late-onset non-atopic patients (the “intrinsic asthma”). Over the past decades, circulating autoantibodies against diverse self-targets (beta-2-adrenergic receptors, epithelial antigens, nuclear antigens, etc.) have been reported and subsequently dismissed to be epiphenomena resulting from a chronic inflammatory condition, primarily due to lack of evidence of causality/pathomechanism. Recent evidence of ‘granulomas’ in the lung biopsies of severe asthmatics, detection of pathogenic sputum autoantibodies against autologous eosinophil proteins (e.g., eosinophil peroxidase) and inadequate response to monoclonal antibody therapies (e.g., subcutaneous mepolizumab) in patients with evidence of airway autoantibodies suggest that the role of autoimmune mechanisms be revisited. In this review, we have gathered available reports of autoimmune responses in the lungs, reviewed the evidence in the context of immunogenic tissue-response and danger-associated molecular patterns, and constructed the possibility of an autoimmune-associated pathomechanism that may contribute to the severity of asthma.

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Endogenous peroxidases in sputum interfere with horse-radish peroxidase-based ELISAs

Cited by 8 publications

References 18 publications

Iron in airway macrophages and infective exacerbations of chronic obstructive pulmonary disease

Iron in airway macrophages and infective exacerbations of chronic obstructive pulmonary disease

Electrochemical detection of uric acid in undiluted human saliva using uricase paper integrated electrodes

Autoimmune Responses in Severe Asthma

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