High‐Throughput, Multiplexed Analysis of 3‐Nitrotyrosine in Individual Proteins

Jin, Hongjun; Zangar, Richard C.

doi:10.1002/0471140856.tx1715s51

Cited by 5 publications

(4 citation statements)

References 26 publications

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“…Regarding 3-NT quantification, there is a wide variety of ELISAbased methods for this purpose, namely indirect, competitive, sandwich-ELISA and ELISA microarrays [68,69]. Table 2 lists some ELISA assays for the analysis of 3-NT in different biological specimens.…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

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3-Nitrotyrosine quantification methods: Current concepts and future challenges

et al. 2016

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Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

“…Regarding microarray-ELISA assays, one of their main advantages is that they use various physically separated capture antibodies (in isolated spots), allowing an efficient way for measuring low levels of the analyte [68].…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

3-Nitrotyrosine quantification methods: Current concepts and future challenges

et al. 2016

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“…For the analysis of total antigen concentrations, custom antibody microarray chips were manufactured and the sandwich ELISA microarray analysis was performed essentially as previously described (Jin and Zangar 2012). For the halotyrosine analyses, the detection antibody was biotinylated BTK-94C.…”

Section: Methodsmentioning

confidence: 99%

A halotyrosine antibody that detects increased protein modifications in asthma patients

Jin

Hallstrand

Daly

et al. 2014

Journal of Immunological Methods

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Airway inflammation has a pathophysiological role in asthma. Eosinophils, which are commonly increased in asthmatic airways, express eosinophil peroxidase and thereby produce hypobromite and bromotyrosine. Bromotyrosine is believed to be a specific marker for eosinophil activity, but developing an antibody against monobromotyrosine, the predominate brominated tyrosine residue found in vivo has proven difficult. We evaluated whether a 3-bromobenozoic acid hapten antigen produced antibodies that recognized halogenated tyrosine residues. Studies with small-molecule inhibitors or brominated or chlorinated protein suggested that a mouse monoclonal antibody (BTK-94C) selectively bound free and protein mono- and dibromotyrosine and, to a lesser degree, chlorotyrosine, and thus was designated a general halotyrosine antibody. We evaluated if this antibody had potential for characterizing human asthma using an enzyme-linked immunosorbent assay (ELISA) microarray platform to examine the halogenation of 23 proteins in three independent sets of sputum samples (52 samples total). In 15 healthy control or asthmatic subjects, ICAM, PDGF and RANTES had greater proportional amounts of halogenation in asthmatic subjects and the halogenation signal was associated with the severity of exercise-induced airway hyperresponsiveness. In 17 severe asthma patients treated with placebo or mepolizumab to suppress eosinophils, drug-related decreases in halogenation were observed with p values ranging from 0.006 to 0.11 for these 3 proteins. Analysis of 20 subjects that either had neutrophilic asthma or were healthy controls demonstrated a broad increase in halotyrosine (possibly chlorotyrosine) in neutrophilic asthmatics. Overall, these results suggest that an ELISA utilizing BTK-94C could prove useful for assessing airway inflammation in asthma patients.

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“…The basic ELISA microarray protocol has been previously described in detail [13]. In brief, 27 capture antibodies (listed in supplementary Table 1) were printed on each chip in quadruplicate spots such that one replicate spot per antibody was printed in each of 4 identical quadrants (Fig.…”

Section: Methodsmentioning

confidence: 99%

Oxidatively modified proteins as plasma biomarkers in breast cancer

Jin

Daly

Marks

et al. 2013

CBM

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BACKGROUND Post-translational protein modifications (PTMs) are increased in breast tumors. OBJECTIVE We explored whether PTMs on proteins secreted by the breast could be detected in plasma and potentially used for the early detection of breast cancer. METHODS We used a custom ELISA microarray platform to measure 4-hydroxynonenal (HNE), glutathione (GSH), nitrotyrosine and halotyrosine adducts in 27 secreted proteins, for a total of 108 candidate biomarkers. Two independent sets human plasma samples were measured, for a total of 160 samples. The results were analyzed for consistent cancer-associated changes across the two sample sets. Plasma samples for both cases and benign controls were collected at the time of tissue diagnosis after referral from a positive screen (such as mammography). The results from both studies were evaluated using ANOVA and t-tests or receiver operator curves (ROC). RESULTS Levels of GSH-modified ceruloplasmin and HNE-modified PDGF were significantly altered in plasma samples from cancer patients relative to benign controls. Healthy controls, which were only included in the first set of samples, were similar to the benign controls for both of these markers. A combination of three glutathionylated proteins had the best area under the ROC curve, with a value of 76%. CONCLUSIONS Specific PTMs in individual proteins may be useful for distinguishing between women with breast cancer and those with benign breast disease. These oxidative changes in plasma proteins may reflect redox changes in breast cancer. Additional studies on oxidative modifications in individual proteins are warranted.

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High‐Throughput, Multiplexed Analysis of 3‐Nitrotyrosine in Individual Proteins

Cited by 5 publications

References 26 publications

3-Nitrotyrosine quantification methods: Current concepts and future challenges

3-Nitrotyrosine quantification methods: Current concepts and future challenges

A halotyrosine antibody that detects increased protein modifications in asthma patients

Oxidatively modified proteins as plasma biomarkers in breast cancer

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