Development and validation of a time-resolved fluorometric immunoassay for screening of antichlamydial activity using a genus-specific europium-conjugated antibody.
The lack of high-throughput assays has limited the screening of new antimicrobials against obligate intracellular bacteria, including chlamydia, which cause a variety of diseases. In this study, a novel technological approach was developed to detect intracellular bacteria using time-resolved fluorometric immunoassay (TR-FIA), and the method was validated for susceptibility testing of Chlamydia pneumoniae. In this cell-based, 96-well plate assay, chlamydial inclusions are labeled with europium-conjugated antibody and quantified as time-resolved fluorometric signals by means of a multilabel counter. To confirm the reliability of the TR-FIA, susceptibilities of C. pneumoniae reference strain Kajaani 7 to a set of antimicrobial agents were determined by the TR-FIA, conventional immunofluorescence staining, and real-time polymerase chain reaction. Minimum inhibitory concentrations measured using the different methods demonstrated good to excellent correlation. Data relating to reproducibility (day-to-day variation 9.0%), as well as to the signal-to-background, signal-to-noise, and Z' values (6.5, 6.9, and 0.4, respectively), showed the suitability of the TR-FIA for screening. By means of dual labeling with sulfornodamine B the cytotoxicity of test compounds could be detected simultaneously with the susceptibility testing. In summary, the TR-FIA is a convenient, reliable, and objective alternative for detecting chlamydia in vitro. By being considerably less labor intensive and offering significantly higher throughput, the TR-FIA is especially suitable for screening of new antichlamydial compounds.
Tammela
Päivi
P
Viikki Drug Discovery Technology Center, Faculty of Pharmacy, P.O. Box 56, University of Helsinki, FIN-00014 Helsinki, Finland.
Alvesalo
Joni
J
Riihimäki
Laura
L
Airenne
Sari
S
Leinonen
Maija
M
Hurskainen
Pertti
P
Enkvist
Kristian
K
Vuorela
Pia
P
D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D000907:Antibodies, Bacterial / Q000737:chemistry* / Q000276:immunology*; D002460:Cell Line; D016993:Chlamydophila pneumoniae / Q000737:chemistry* / Q000254:growth & development / Q000276:immunology*; D005063:Europium / Q000737:chemistry*; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; D000907:Antibodies, Bacterial; D005063:Europium