A Wide-Field Fluorescence Microscope Extension for Ultrafast Screening of One-Bead One-Compound Libraries Using a Spectral Image Subtraction Approach.

The increasing involvement of academic institutions and biotech companies in drug discovery calls for cost-effective methods to identify new bioactive molecules. Affinity-based on-bead screening of combinatorial one-bead one-compound libraries combines a split-mix synthesis design with a simple protein binding assay operating directly at the bead matrix. However, one bottleneck for academic scale on-bead screening is the unavailability of a cheap, automated, and robust screening platform that still provides a quantitative signal related to the amount of target protein binding to individual beads for hit bead ranking. Wide-field fluorescence microscopy has long been considered unsuitable due to significant broad spectrum autofluorescence of the library beads in conjunction with low detection sensitivity. Herein, we demonstrate how such a standard microscope equipped with LED-based excitation and a modern CMOS camera can be successfully used for selecting hit beads. We show that the autofluorescence issue can be overcome by an optical image subtraction approach that yields excellent signal-to-noise ratios for the detection of bead-associated target proteins. A polymer capillary attached to a semiautomated bead-picking device allows the operator to efficiently isolate individual hit beads in less than 20 s. The system can be used for ultrafast screening of >200,000 bead-bound compounds in 1.5 h, thereby making high-throughput screening accessible to a wider group within the scientific community.

Quantitative detection of Porphyromonas gingivalis fimA genotypes in dental plaque.

We developed quantitative fimA genotype assays and applied them in a pilot study investigating the fimbrial genotype distribution of Porphyromonas gingivalis in European subjects with or without chronic periodontitis. P. gingivalis was found in 71% and 9% of the samples from patients and healthy subjects, respectively. Enumeration of total P. gingivalis cell numbers by polymerase chain reaction and immunofluorescence showed excellent correspondence (r = 0.964). 73% of positive samples contained multiple fimA genotypes, but generally one genotype predominated by one to three orders of magnitude. Genotype II predominated in 60% of the samples. Genotype IV occurred with similar prevalence (73%) as genotype II but predominated in only 20% of the samples. Genotypes I, III and V were of much lower prevalence and cell densities of the latter two remained sparse. Our results suggest marked differences among the fimA genotypes' ability to colonize host sites with high cell numbers.