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Measurement of serum antibody to subgingival bacterial species has been useful in discriminating possible periodontal pathogens and in assessing the host's immune response to subgingival species. An immunoassay system was developed to measure the level of serum antibody to multiple subgingival species in multiple serum samples on a single nitrocellulose membrane. The principle steps of the assay are the following: 1) binding of antigens from bacterial preparations and protein A in parallel lanes on nitrocellulose membranes; 2) incubation of known concentrations of human immunoglobulin as well as various dilutions of serum from patients in lanes perpendicular to the antigen lanes; 3) incubation of the membrane with a peroxidase-conjugated second antibody; 4) detection of positive reactions by enhanced chemiluminescence. Emitted light was captured on a photographic film in which the positive reactions appeared as squares at the intersections of antigens with appropriate antibody. Antibody was quantified using a laser densitometer to compare the signal intensity of unknown samples with the ones generated by known amounts of human immunoglobulin captured on the same membrane. The assay permitted simultaneous screening and/or quantification of antibody in as many as 45 serum samples against up to 45 bacterial species. The mean and standard error of the coefficients of variation for replicates within an assay averaged 7.3 +/- 2.3%. Coefficients of variation of the assay run on different days for serum antibody to a range of subgingival species averaged 10.1 +/- 2.1%. Checkerboard immunoblotting is a simple and rapid immunoassay to permit quantification and/or screening of antibody to multiple subgingival species or antigens in multiple serum samples.

The purpose of this investigation was to compare the levels of serum IgG2, the frequency of detection of Gm(23)-negative allotype and frequency of detection of FcgammaRIIa and FcgammaRIIIb receptor haplotypes in 32 refractory, 54 successfully treated and 27 periodontally healthy individuals. Refractory subjects showed mean full mouth attachment loss and/or >3 sites with attachment loss >2.5 mm within 1 year after both scaling and root planing, and surgery plus systemically administered tetracycline. Successfully treated subjects showed mean attachment level gain and no sites with attachment loss >2.5 mm 1 year post-therapy. Periodontally healthy subjects exhibited no pocket depth or attachment level >3 mm, and no evidence of progressing disease during 1 year of monitoring. Blood was obtained from each subject at baseline. Serum IgG2 and Gm(23) allotype were determined using radial immunodiffusion. DNA was extracted from whole blood and the FcgammaR genotypes determined using PCR and allele specific oligonucleotide probes. Significance of differences among clinical groups were sought using the Kruskal-Wallis or chi-square tests. Associations between 2 or more variables were tested using regression analysis. Refractory subjects exhibited higher mean attachment loss and pocket depth than successfully treated or periodontally healthy subjects. Smoking status did not differ significantly among groups. No significant differences in serum IgG2 levels and frequency of detection of Gm(23)-negative allotype were observed among the clinical groups. Serum IgG2 level was positively associated with the number of serum antibody responses to subgingival species (r=0.51, p<0.001). Subjects with the Gm(23)-negative allotype exhibited lower mean levels of serum IgG2 (3.06+/-0.3 versus 3.9+/-0.2, p<0.01) and mean number of serum antibodies to subgingival species (17.7+/-1.7 versus 23.3+/-1.4, p<0.05) than allotype positive individuals. No significant differences in FcgammaR haplotype distribution were observed among the 3 clinical groups. Associations of serum IgG2 level, Gm(23) allotype, FcgammaRIIa and FcgammaRIIIb receptor haplotypes and smoking status were weakly related or not related to clinical status. This lack of relationship may have been due to a reality of no relationship, or the inadvertent pooling of subjects where these factors were of primary importance with subjects in whom these factors played a less important role.

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The effect of short-chain fatty acids on the phagocytic activity of human alveolar macrophages and neutrophils was investigated. These acids, butyric, propionic, and succinic, are produced by anaerobic bacteria. The results indicate that phagocytosis of Staphylococcus aureus by human lung phagocytes is strongly inhibited by the end products of anaerobic catabolism and support the hypothesis that the antiphagocytic activity present in the supernatants of anaerobic cultures may be dependent on the presence of short-chain fatty acids.