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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.

A chemical method for labeling of oligonucleotide probes with europium chelates is presented. A modified deoxycytidine phosphoramidite is used to introduce multiple reactive amino groups to the oligonucleotide during the synthesis phase. Upon deprotection and purification of the modified oligonucleotide, an isothiocyanate derivative of a stable Eu chelate is reacted with the primary amino groups. The labeling technology enables the coupling of a high number of Eu chelates to a single probe. The melting temperatures and hybridization efficiencies of the oligonucleotides are not significantly altered by the labeling process. However, hybridization kinetics of the oligonucleotides are affected by the introduction of multiple modified deoxycytidine residues. In a solid-phase hybridization assay up to 10(7) target molecules can be detected.

We describe a novel assay for detection of point mutations. The method combines the specificity and sensitivity of the polymerase chain reaction (PCR) and allele-specific oligonucleotides (ASO) with highly sensitive time-resolved fluorometry. ASO probes differing by a single base substitution and labeled with europium (Eu) chelates were hybridized in solution simultaneously with a biotinylated oligomer to a PCR-amplified nucleic acid fragment. The hybrids formed were then collected onto streptavidin-coated microtitration wells. Subsequently, the hybrids were washed under stringent conditions and the remaining ASO probe was measured in a time-resolved fluorometer. We discuss the strategy underlying the design of the Eu-labeled ASO probes for the solution hybridization assay. The method was applied to the detection of the Z-mutation in the alpha 1-antitrypsin gene. Evaluation of whole-blood samples spotted on Guthrie cards demonstrated successful accuracy of the method.

The polymerase chain reaction (PCR) has many potential applications in the field of DNA probe diagnostics. Here we describe a method that utilizes PCR and time-resolved fluorometry (TRF) for the detection of specific target DNA. First the DNA segment to be detected is amplified according to standard procedures. Then a pair of europium (Eu3+) and biotin-labelled primers nested within the amplified fragment is incorporated in a few additional PCR cycles. Thus amplified DNA fragments are generated that contain an affinity label (biotin) and a detectable label (europium). The doubly-labelled amplified DNA fragments are collected onto streptavidin coated microtitration strips and the bound Eu3+ is measured in a time-resolved fluorometer. We show here the application of this method to the detection of HIV-1 DNA. As few as five copies of HIV-1 DNA could readily be detected using this assay. The method described here is sensitive, rapid and easy to employ. In addition it lends itself to automation.

A chemical method for labelling DNA with a europium chelate is presented. First, primary aliphatic amino groups are introduced onto DNA in a transamination reaction. The transamination reaction is altered by adjusting temperature and duration of the reaction. Subsequently, the modified DNA is reacted with an isothiocyanate derivative of a Eu chelate. The optimum amount of Eu chelates on a DNA probe is 4-8% of total nucleotides. There is a decrease of 0.7 degrees C in the melting temperature of DNA for each incorporated Eu chelate on 100 bases. Hybridization efficiency is lowered by the introduction of Eu chelates but this effect can be partly overcome by using high DNA probe concentrations. The detection limit of the Eu-labelled probe is 0.15 attomoles of target DNA in a mixed-phase hybridization assay on microtitration wells. In addition to high sensitivity the Eu-labelled probes offer convenience in use and results which are quantitative and easy to interpret.