A His6-SUMO-eXact tag for producing human prepro-Urocortin 2 in Escherichia coli for raising monoclonal antibodies

Liew, Oi Wah; Ang, Cui Xia; Peh, Yu Pei; Chong, Pek Ching Jenny; Ng, Yan Xia; Hwang, Le-Ann; Koh, Xin Yu; Yip, Yin Mun; Liu, Wei; Richards, A. Mark

doi:10.1016/j.jim.2013.11.015

Cited by 12 publications

(10 citation statements)

References 46 publications

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“…The E. coli host strain BL21 (DE3) trxB (Novagen, Merck Millipore, Darmstadt, Germany) was used for the expression of the three recombinant Trxp53mutant constructs. Mini-scale expression studies to determine the solubility of the protein were performed as described previously (Liew et al, 2014). For large-scale purification, recovery of soluble and insoluble peptides was done by native and denaturing IMAC with integrated on-column refolding into phosphate buffer, respectively, and was performed as described previously (Liew et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

Monoclonal Antibodies against Specific p53 Hotspot Mutants as Potential Tools for Precision Medicine

et al. 2018

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The large number of mutations identified across all cancers represents an untapped reservoir of targets that can be useful for therapeutic targeting if highly selective, mutation-specific reagents are available. We report here our attempt to generate such reagents: monoclonal antibodies against the most common R175H, R248Q, and R273H hotspot mutants of the tumor suppressor p53. These antibodies recognize their intended specific alterations without any cross-reactivity against wild-type (WT) p53 or other p53 mutants, including at the same position (as exemplified by anti-R248Q antibody, which does not recognize the R248W mutation), evaluated by direct immunoblotting, immunoprecipitation, and immunofluorescence methods on transfected and endogenous proteins. Moreover, their clinical utility to diagnose the presence of specific p53 mutants in human tumor microarrays by immunohistochemistry is also shown. Together, the data demonstrate that antibodies against specific single-amino-acid alterations can be generated reproducibly and highlight their utility, which could potentially be extended to therapeutic settings.

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Section: Methodsmentioning

confidence: 99%

Monoclonal Antibodies against Specific p53 Hotspot Mutants as Potential Tools for Precision Medicine

et al. 2018

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“…The mouse monoclonal antibody (mAb 2D7) directed against human NT-proUcn2 described previously (18 ) was used as the capture antibody. The assay calibrator was a bacterially produced recombinant protein (TrxUCN2pro) comprising thioredoxin fused to an His 6 tag and NT-proUcn2 with a predicted mass of 23564 Da (see Fig.…”

Section: Antibodiesmentioning

confidence: 99%

“…Purified TrxUCN2pro was diluted to obtain calibrators yielding equivalent NT-proUcn2 concentrations between 23.8 -572 ng/L (approximately 4.3-102 pmol/L; NT-proUcn2 molecular mass of 5585 Da). Test proteins for evaluating assay cross-reactivity to bovine, mouse, and rat NT-proUcn2 were produced as thioredoxin fusions by cloning strategies described previously (18 ). His 6 -SUMO-eXact tag fused to prepro-Urocortin 2, as described previously, was used as antigen for rabbit polyclonal antibody production (18 ).…”

Section: Antibodiesmentioning

confidence: 99%

“…Purified 2D7 was immobilized on a GLC chip as described previously (18 ) and tested against 100 nmol/L of thioredoxin fusions of human, bovine, rat, and mouse NT-proUcn2.…”

Section: Validation Studiesmentioning

confidence: 99%

“…Ucn2 prohormone and postulated the value of measuring endogenous proUcn2 as a surrogate measure of mature Ucn2 (18 ). The few reports to date on plasma Ucn2 in clinical cardiovascular disease offer implausibly discrepant values spread over a thousand-fold range for which little assay validation is available.…”

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High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure

et al. 2016

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BACKGROUND Urocortin 2 (Ucn2) has powerful hemodynamic, renal, and neurohormonal actions and likely participates in normal circulatory homeostasis and the compensatory response to heart failure (HF). A validated assay for endogenous circulating Ucn2 would facilitate investigations into Ucn2 physiology and elucidate its derangement and potential as a biomarker in heart disease. METHOD We developed a chemiluminescence-based sandwich ELISA to measure plasma N-terminal (NT)-proUcn2 in non-HF patients (control; n = 160) and HF patients with reduced (HFREF; n = 134) and preserved (HFPEF; n = 121) left ventricular ejection fraction (LVEF). RESULTS The ELISA had a limit of detection of 8.47 ng/L (1.52 pmol/L) and working range of 23.8–572 ng/L. Intra- and interassay CV and total error were 4.8, 16.2, and 17.7%, respectively. The median (interquartile range) plasma NT-proUcn2 concentration in controls was 112 (86–132) ng/L. HFREF, HFPEF, and all HF plasma concentrations were significantly increased [117 (98–141) ng/L, P = 0.0007; 119 (93–136) ng/L, P = 0.0376, and 119 (97–140) ng/L, P = 0.001] compared with controls but did not differ significantly between HFREF and HFPEF. NT-proUcn2 was modestly related to age (r = 0.264, P = 0.001) and cardiac troponin T (r = 0.258, P = 0.001) but not N-terminal pro-B-type natriuretic peptide, body mass index, LVEF, or estimated glomerular filtration rate. On multivariate analysis, plasma NT-proUcn2 was independently and inversely related to 2-year mortality in HF. CONCLUSIONS The validated ELISA measured human NT-proUcn2 in plasma and showed modest but significant increases in HF patients compared with controls. In HF, the unusual inverse relationship between plasma NT-proUcn2 and 2-year mortality portends potential prognostic value but requires further corroboration.

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Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies

Ling,

Lilyanna,

et al. 2024

Cell. Mol. Life Sci.

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Neprilysin (NEP) is an emerging biomarker for various diseases including heart failure (HF). However, major inter-assay inconsistency in the reported concentrations of circulating NEP and uncertainty with respect to its correlations with type and severity of disease are in part attributed to poorly characterized antibodies supplied in commercial ELISA kits. Validated antibodies with well-defined binding footprints are critical for understanding the biological and clinical context of NEP immunoassay data. To achieve this, we applied in silico epitope prediction and rational peptide selection to generate monoclonal antibodies (mAbs) against spatially distant sites on NEP. One of the selected epitopes contained published N-linked glycosylation sites at N285 and N294. The best antibody pair, mAb 17E11 and 31E1 (glycosylation-sensitive), were characterized by surface plasmon resonance, isotyping, epitope mapping, and western blotting. A validated two-site sandwich NEP ELISA with a limit of detection of 2.15 pg/ml and working range of 13.1–8000 pg/ml was developed with these mAbs. Western analysis using a validated commercial polyclonal antibody (PE pAb) and our mAbs revealed that non-HF and HF plasma NEP circulates as a heterogenous mix of moieties that possibly reflect proteolytic processing, post-translational modifications and homo-dimerization. Both our mAbs detected a ~ 33 kDa NEP fragment which was not apparent with PE pAb, as well as a common ~ 57–60 kDa moiety. These antibodies exhibit different affinities for the various NEP targets. Immunoassay results are dependent on NEP epitopes variably detected by the antibody pairs used, explaining the current discordant NEP measurements derived from different ELISA kits.

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A His6-SUMO-eXact tag for producing human prepro-Urocortin 2 in Escherichia coli for raising monoclonal antibodies

Cited by 12 publications

References 46 publications

Monoclonal Antibodies against Specific p53 Hotspot Mutants as Potential Tools for Precision Medicine

Monoclonal Antibodies against Specific p53 Hotspot Mutants as Potential Tools for Precision Medicine

High-Sensitivity Sandwich ELISA for Plasma NT-proUcn2: Plasma Concentrations and Relationship to Mortality in Heart Failure

Multiple circulating forms of neprilysin detected with novel epitope-directed monoclonal antibodies

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