Monoclonal antibodies to lipopolysaccharide of four oral bacteria associated with periodontal disease.

Periodontal disease is a common inflammatory disease which erodes the supporting structures of the teeth, and is initiated by a subgingival infection with selected Gram-negative bacteria. Monoclonal antibodies (mAb) to lipopolysaccharide (LPS) of four periodontal pathogens, A. actinomycetemcomitans, P. intermedia, F. nucleatum and P. gingivalis were examined for specificity and their ability to bind these pathogens in a particle concentration fluorescence immunoassay (PCFIA). The mAb selected were specific for their homologous bacteria and when tested against a large battery of other bacteria, including 16 genera and 46 species, were found not to cross-react with heterologous species. When each of the mAb was challenged with 40 or more homologous freshly isolated bacteria, more than 90% were positive. Non-cellular antigens in the form of soluble LPS and extracellular vesicles were examined for their ability to bind to assay components and alter the apparent results of the assay. LPS was found to have potential as an interfering agent if bound to assay components prior to sample treatment, but this non-specific binding was significantly reduced when a surfactant was added to the buffers. Extracellular vesicles had no significant effect on the estimation of P. gingivalis by the assay.

Bacterial concentration fluorescence immunoassay (BCFIA) for the detection of periodontopathogens in plaque.

A bacterial concentration fluorescence immunoassay (BCFIA) was developed to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilized fluorescent-tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected Gram-negative bacteria. Microorganisms identified in plaque using the BCFIA included Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. The immunoassay procedure involved combining a patient's plaque sample with a species-specific fluorescein isothiocyanate-labeled MAb and then incubating the mixture in a specialized microtiter plate allowing the MAb to bind to its homologous bacteria. Bound and unbound fluorescent-tagged MAbs were separated by filtration and total bound bacterial fluorescence was determined with a fluorimeter. The relative number of a bacterial species in a given plaque sample was estimated by reference to a standard curve carried through the BCFIA. The BCFIA had a lower detection limit of near 10(4) specific bacterial cells in a mixed bacterial preparation or plaque sample. When compared to cultivable flora procedures in detecting the 4 periodontopathogens, the BCFIA had high levels of statistical sensitivity, 97% to 100%, while statistical specificity ranged between 57% and 92%. There was a 71% to 82% agreement between BCFIA and DNA probe methodology in detecting periodontopathogens in plaque. The BCFIA, when compared to cultivable flora, offers the advantage of evaluating both live and dead bacterial cells in plaque. This may in part, if not fully, explain the lower specificity values of the BCFIA when compared to cultivable flora. Screening plaque samples for periodontopathic bacteria is considerably faster and results in a greater frequency of detection with BCFIA than cultivable flora based methods.