Development and validation of a time-resolved fluorometric immunoassay for screening of antichlamydial activity using a genus-specific europium-conjugated antibody

Tammela, Päivi; Alvesalo, Joni; Riihimäki, Laura; Airenne, Sari; Leinonen, Maija; Hurskainen, Pertti; Enkvist, Kristian; Vuorela, Pia

doi:10.1016/j.ab.2004.06.012

Cited by 17 publications

(15 citation statements)

References 48 publications

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“…The TR-FIA protocol has been described in detail elsewhere [18]. Briefly, 80%-90% confluent HL cell layers were infected in 96-well plates (6 ϫ 10 4 cells/well) with 1.2 ϫ 10 5 purified C. pneumoniae EBs/well 16 h after transfection.…”

Section: Methodsmentioning

confidence: 99%

Microarray Analysis of aChlamydia pneumoniae–Infected Human Epithelial Cell Line by Use of Gene Ontology Hierarchy

et al. 2008

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Chlamydia pneumoniae, a gram-negative obligate intracellular bacterium, is a common cause of upper and lower respiratory tract infections worldwide. Persistent C. pneumoniae infections have been linked to chronic disease processes, such as atherosclerosis. In the present study, we examined gene expression changes in the human epithelial cell line at different stages of acute C. pneumoniae infection and used gene ontology annotation, along with single-gene analysis, to select a small group of target genes that could possibly play a key role in C. pneumoniae infection. Selected genes were silenced using small interfering RNA, and the effect of silencing on the number of C. pneumoniae inclusions was measured by time-resolved fluorometric immunoassay. The greatest reduction in the number of C. pneumoniae inclusions was due to the silencing of the gene coding for the transcription factor early growth response 1, which decreased the number of inclusions by 38.6%.

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Section: Methodsmentioning

confidence: 99%

Microarray Analysis of aChlamydia pneumoniae–Infected Human Epithelial Cell Line by Use of Gene Ontology Hierarchy

et al. 2008

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“…The potency (50% inhibitory concentration) value of mycophenolic acid was in the high nanomolar range. With a calculated MIC 90 of 1 μM, mycophenolic acid has a higher potency against C. pneumoniae than ofloxacin, for which an MIC 90 value of 1.38 μM has been reported with the same strain of C. pneumoniae , K7, and the same detection method . Mycophenolic acid has been found to display antimicrobial activities, and it has been approved as an immunosuppressant drug to prevent organ transplant rejection.…”

Section: Resultsmentioning

confidence: 99%

Identification of Privileged Antichlamydial Natural Products by a Ligand-Based Strategy

et al. 2017

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The obligate intracellular pathogen Chlamydia pneumoniae remains a difficult target for antimicrobial therapy. Owing to the permeability barrier placed by bacterial and host vacuolar membranes, as well as the propensity of the bacterium for persistent infections, treatment failures are common. Despite the urgent need for new antichlamydial compounds, their discovery is challenged by the technically demanding assay procedures and lack of validated targets. An alternative strategy of using naturally occurring compounds and their derivatives against C. pneumoniae is presented. The strategy consists of the application of ligand-based virtual screening to a natural product library of 502 compounds with the ChemGPS-NP chemography tool followed by in vitro antichlamydial assays. The reference set used for the 2D similarity search was constructed of 19 known antichlamydial compounds of plant origin. Based on the similarity screen, 53 virtual hits were selected for in vitro testing. Six compounds (leads) were identified that cause ≥50% C. pneumoniae growth inhibition and showed no impact on host cell viability. The leads fall into completely new antichlamydial chemotypes, one of them being mycophenolic acid (IC value 0.3 μM). The outcome indicates that using this flipped, target-independent strategy is useful for facilitating the antimicrobial lead discovery against challenging microbes.

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“…However, this requires that the cultures are uniform and the cells have grown to confluency. More complicated automated time‐resolved fluoroimmunoassay utilizing Europium‐labeled anti‐LPS antibodies for staining chlamydial inclusions has earlier been suggested for drug sensitivity studies (10).…”

Section: Resultsmentioning

confidence: 99%

Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real‐time PCR, immunofluorescence and ELISA methods

Poikonen

Lajunen

Silvennoinen-Kassinen

et al. 2009

APMIS

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Chlamydia pneumoniae is an intracellular gram-negative bacterium, which replicates only in eukaryotic cells. Quantification of C. pneumoniae in cell culture is needed when studying e.g. the effect of drugs or host cell factors on infectivity and replication. Conventionally, this has been performed by immunofluorescence staining and microscopic counting of chlamydial inclusions. However, this method is usable only if the cell numbers do not fluctuate in cell culture vials and the inclusions are uniform. In macrophages, inclusions are often aberrant, their sizes vary, and multiple inclusions are also seen. Therefore, methods are needed to quantify exact amounts of C. pneumoniae in cells. Here, we describe a new method based on the real-time PCR quantification of chlamydial genomes adjusted to the number of human genomes in cultures. In human epithelial (HL) cell cultures, the C. pneumoniae inclusion numbers and the ratio of C. pneumonia genomes/human genome (Cpn/Hum) correlated significantly (r = 0.978, p < 0.001); thus with HL cells, both methods are usable. However, in macrophage cultures, the correlation was weaker (r = 0.133, p = 0.036) and we recommend PCR quantification for exact measurements.

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Development and validation of a time-resolved fluorometric immunoassay for screening of antichlamydial activity using a genus-specific europium-conjugated antibody

Cited by 17 publications

References 48 publications

Microarray Analysis of aChlamydia pneumoniae–Infected Human Epithelial Cell Line by Use of Gene Ontology Hierarchy

Microarray Analysis of aChlamydia pneumoniae–Infected Human Epithelial Cell Line by Use of Gene Ontology Hierarchy

Identification of Privileged Antichlamydial Natural Products by a Ligand-Based Strategy

Quantification of Chlamydia pneumoniae in cultured human macrophages and HL cells: comparison of real‐time PCR, immunofluorescence and ELISA methods

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