RESEARCH ARTICLE Anti-HmuY Antibodies Specifically Recognize Porphyromonas gingivalis HmuY Protein but Not Homologous Proteins in Other

Given the emerging evidence of an association between periodontal infections and system-ic conditions, the search for specific methods to detect the presence of P. gingivalis, a princi-pal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY pro-tein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tan-nerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blot-ting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to puri-fied HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. inter-media and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gin-givalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium