A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma.
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ABSTRACT:
SUBMITTER: Goiffon RJ
PROVIDER: S-EPMC4347050 | biostudies-other | 2015 Feb
REPOSITORIES: biostudies-other
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Project description:BackgroundEnzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.Principal findingsWe found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.ConclusionWe validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.
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Project description:Bacillus cereus is a foodborne pathogen causing emesis and diarrhea in those affected. It is assumed that the non-hemolytic enterotoxin (Nhe) plays a key role in B. cereus induced diarrhea. The ability to trace Nhe activity is important for food safety. While assays such as PCR and ELISA exist to detect Nhe, those methods cannot differentiate between active and inactive forms of Nhe. The existing rabbit ileal loop bioassay used to detect Nhe activity is ethically disfavored because it uses live experimental animals. Here we present a custom built low-cost CCD based luminometer and applied it in conjunction with a cell-based assay using Vero cells transduced to express the luciferase enzyme. The activity of Nhe was measured as its ability to inhibit synthesis of luciferase as quantified by reduction of light emission by the luciferase reaction. Emitted light intensity was observed to be inversely proportional to Nhe concentration over a range of 7 ng/ml to 125 ng/ml, with a limit of detection of 7 ng/ml Nhe.
| S-EPMC7527251 | biostudies-literature
Project description:In traditional Chinese medicine herbs (TCM), including Radix Salviae Miltiorrhizae (Danshen), Radix Puerariae Lobatae (Gegen), Radix Angelicae Sinensis (Danggui), and Rhizoma Chuanxiong (Chuanxiong) are widely used for the prevention and treatment of cardiovascular diseases and also often co-administered with Western drugs as a part of integrative medicine practice. Carboxylesterase 1 (CES1) plays a pivotal role in the metabolisms of pro-drugs. Since (S)-2-(2-(6-dimethylamino)-benzothiazole)-4,5-dihydro-thiazole-4-carboxylate (NLMe) has recently been identified by us as a selective CES1 bioluminescent sensor, we developed a rapid method using this substrate for the direct measurement of CES1 activity in rats. This bioluminescence assay was applied to determine CES1 activity in rat tissues after a two-week oral administration of each of the four herbs noted above. The results demonstrated the presence of CES1 enzyme in rat blood and all tested tissues with much higher enzyme activity in the blood, liver, kidney and heart than that in the small intestine, spleen, lung, pancreas, brain and stomach. In addition, the four herbs showed tissue-specific effects on rat CES1 expression. Based on the CES1 biodistribution and its changes after treatment in rats, the possibility that Danshen, Gegen and Danggui might alter CES1 activities in human blood and kidney should be considered. In summary, a selective and sensitive bioluminescence assay was developed to rapidly evaluate CES1 activity and the effects of orally administered TCMs in rats.
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Project description:Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to target reactive oxygen and nitrogen species during polymorphonuclear neutrophil inflammatory burst following injury. Here, we review the following 1) the broad effect of myeloperoxidase on normal physiology, 2) the difference between myeloperoxidase and other peroxidases, 3) the current optical probes available for use as surrogates for direct measures of myeloperoxidase-derived oxidants, and 4) the range of preclinical options for imaging myeloperoxidase accumulation at sites of inflammation in mice. We also stress the advantages and drawbacks of each of these methods, the pharmacokinetic considerations that may limit probe use to strictly cell cultures for some reactive oxygen and nitrogen species, rather than in vivo utility as indicators of myeloperoxidase function. Taken together, our review should shed light on the fundamental rational behind these techniques for measuring myeloperoxidase activity and polymorphonuclear neutrophil response after injury toward developing safe myeloperoxidase inhibitors as potential therapy for chronic obstructive pulmonary disease and rheumatoid arthritis.
| S-EPMC6608033 | biostudies-literature
Project description:Prions are infectious proteins that cause fatal neurodegenerative diseases. Because astrocytic gliosis marked by the deposition of fibrils composed of GFAP is a prominent feature of prion disease, we asked whether GFAP might be used as a surrogate marker for prions. To interrogate this posit, we inoculated prions into transgenic (Tg) mice expressing luciferase (luc) under the GFAP gene (Gfap) promoter, denoted Tg(Gfap-luc) mice. Weekly noninvasive, bioluminescence imaging (BLI) detected an increase in light emitted from the brains of Tg(Gfap-luc) mice at approximately 55 d after inoculation and approximately 62 d before neurologic deficits appeared. To determine whether BLI could be used as a proxy bioassay for prion infectivity, we performed endpoint titrations of prions in Tg(Gfap-luc) mice. BLI bioassays were as or more sensitive than those determined by the onset of neurological dysfunction, and were completed in approximately half the time. Our studies argue that BLI is likely to be a suitable surrogate for measuring prion infectivity, and might be useful in the study of Tg mouse models for other neurodegenerative illnesses.
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Project description:BACKGROUND:Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a glycosylphosphatidylinositol (GPI)-anchored protein of capillary endothelial cells, transports lipoprotein lipase to the capillary lumen and is essential for the lipolytic processing of triglyceride-rich lipoproteins. OBJECTIVE:Because some GPI-anchored proteins have been detected in plasma, we tested whether GPIHBP1 is present in human blood and whether GPIHBP1 deficiency or a history of cardiovascular disease affected GPIHBP1 circulating levels. METHODS:We developed 2 monoclonal antibodies against GPIHBP1 and used the antibodies to establish a sandwich enzyme-linked immunosorbent assay (ELISA) to measure GPIHBP1 levels in human blood. RESULTS:The GPIHBP1 ELISA was linear in the 8 to 500 pg/mL range and allowed the quantification of GPIHBP1 in serum and in pre- and post-heparin plasma (including lipemic samples). GPIHBP1 was undetectable in the plasma of subjects with null mutations in GPIHBP1. Serum GPIHBP1 median levels were 849 pg/mL (range: 740-1014) in healthy volunteers (n = 28) and 1087 pg/mL (range: 877-1371) in patients with a history of cardiovascular or metabolic disease (n = 415). There was an extremely small inverse correlation between GPIHBP1 and triglyceride levels (r = 0.109; P < .0275). GPIHBP1 levels tended to be slightly higher in patients who had a major cardiovascular event after revascularization. CONCLUSION:We developed an ELISA for quantifying GPIHBP1 in human blood. This assay will be useful to identify patients with GPIHBP1 deficiency and patients with GPIHBP1 autoantibodies. The potential of plasma GPIHBP1 as a biomarker for metabolic or cardiovascular disease is yet questionable but needs additional testing.
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Project description:A radioimmunoassay for myeloperoxidase was established with the use of affinity-purified anti-(human myeloperoxidase) immunoglobulins. By the use of ion-exchange followed by immunoaffinity chromatography a preparation of immunoreactive, catalytically active myeloperoxidase was obtained from fresh human plasma. In non-denaturing gel electrophoresis, the plasma preparation showed about four catalytically active components of mobility very similar to that of the granulocyte enzyme. SDS/polyacrylamide-gel electrophoresis combined with protein blotting showed that the two polypeptides of strongest antigenicity in the plasma preparation corresponded in Mr to the large and the small subunits of the granulocyte enzyme. In addition, the plasma preparation contained a higher-Mr immunoreactive polypeptide, possibly a precursor form of the enzyme, together with another of Mr similar to that of the large subunit of eosinophil peroxidase.
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Project description:Nucleotide excision repair (NER) excises bulky DNA lesions induced by mutagens and carcinogens. The repair process includes recognition of DNA damage, excision of a short patch of nucleotides containing the damaged base, re-synthesis of a new DNA strand and ligation of the nicks to restore the sequence integrity. Mutation or aberrant transcription of NER genes reduces repair efficiency and results in the accumulation of mutations that is associated with the development of cancer. Here we present a rapid, sensitive and quantitative assay to measure NER activity in human cells, which we term the Oligonucleotide Retrieval Assay (ORA). We used oligonucleotide constructs containing the UV-damaged adduct, cyclobutane pyrimidine dimer (CPD), to transfect human cells, and retrieved the oligonucleotides for quantification of the repaired, CPD-free DNA by real-time quantitative PCR. We demonstrate that ORA can quantify the extent of NER in diverse cell types, including immortalized, primary and stem-like cells.
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