Monoclonal antibodies produced against lipopolysaccharide from fimA Type II Porphyromonas gingivalis.

An important periodontal pathogen, Porphyromans gingivalis strains are classified into six genotypes (types I-V and Ib), based on the genotype of the fimbriae A (fimA). Among the genotypes, fimA type II strains are thought to be most strongly related to advanced periodontitis. To develop passive immunotherapy, over 300 hybridoma clones were constructed through immunization of cell extracts of fimA type II strain P. gingivalis TDC60 using hybridoma technology. Among these clones, 15 MAbs recognized TDC60 lipopolysaccharide (LPS) with an individual ladder-like structure by Western blot analysis. Further Western blotting of the 15 MAbs against LPS from TDC60, FDC381 (fimA type I), and W83 (fimA type IV) of P. gingivalis and Escherichia coli was carried out. None of these MAbs recognized E. coli LPS, and divided into at least three different Western blot patterns. To confirm the specificity to LPS, three clones were selected and competition assays were carried out using TDC60 LPS. All three MAbs reduced the reactivity against TDC60 LPS after absorption of the LPS in a dose-dependent manner. These findings suggest that MAbs recognizing different epitopes of P. gingivalis LPS were successfully constructed, and these MAbs may be useful in neutralizing P. gingivalis infection.

Egg yolk-derived immunoglobulin (IgY) against Porphyromonas gingivalis 40-kDa outer membrane protein inhibits coaggregation activity.

The anaerobic bacterium Porphyromonas gingivalis, a major pathogen in periodontitis, aggregates with a number of oral bacteria to form dental plaque, which is important for its colonization. We previously cloned the gene coding the 40-kDa outer membrane protein (OMP) of P. gingivalis 381 and produced large amounts of the recombinant (r) protein. Affinity-purified rabbit antiserum against r40-kDa OMP effectively inhibited the coaggregation activity of P. gingivalis to oral bacteria, thus 40-kDa OMP was thought to be an important coaggregation factor of P. gingivalis. Further, since it is conserved among many P. gingivalis strains, this coaggregation factor may be an effective target for passive immunotherapy against P. gingivalis infection. Recently, passive immunization approaches using a specific antibody produced from hen egg yolk (IgY) have been developed for oral infectious diseases, and shown to be convenient and economic. In the present study, we immunized hens intramuscularly with r40-kDa OMP and obtained highly purified IgY from the egg yolks. The purified IgY specifically recognized r40-kDa OMP and also reacted with a functional coaggregation-associated domain peptide of 40-kDa OMP. Our results demonstrated that a ratio of purified IgY as low as 2.5 microg/150 microl significantly inhibited the coaggregation of P. gingivalis with Streptococcus gordonii, which was verified by a visual coaggregation assay and radioactivity-based quantitative micro-coaggregation assay. We concluded anti-r40-kDa OMP IgY may be useful for passive immunization against periodontal diseases caused by P. gingivalis infection.