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The purpose of this study was to assess the recovery of certain proteins from paper strips used for sampling of gingival crevicular fluid (GCF). During a period of 60 min, proteins were eluent in isotonic phosphate-buffered saline, pH 7.4, without detergent. This eluent did not lyse neutrophils applied to the strips, which might otherwise contribute intracellular substances-, e.g., elastase to the GCF samples. The proteins applied (MW 12-725 kD) were satisfactorily recovered (about 90%). However, free neutrophil elastase, unlike elastase complexed with alpha-2-macroglobulin, could not be recovered from the paper strips. Strips made of polyvinylidene difluoride (Durapore) were also tested and 31% of the applied pure elastase was recovered from them. A comparison of measurements made with a Periotron 6000 on Periopaper and Durapore strips, showed that the values obtained with Durapore were too low for accurate measurements of small volumes of fluid. On the whole, when the elution was made in isotonic buffers at neutral pH and without detergents, the recovery of most proteins was satisfactory and reproducible, and independent of MW in the tested range. However, the study also showed poor recovery of uncomplexed elastase.

The Techniques presently available for counting crevicular polymorphonuclear leucocytes (CPMNs) require special equipment and are only semiquantitative. The present new and simple method is based on the property that Durapore strips have to harvest a maximum number of crevicular cells and to release them when shaken in a suitable buffer. Durapore strips were inserted into periodontal crevices or pockets, shaken in PBS and the liberated PMNs counted in a Neubauer chamber. The validity of the method was ascertained in 5 experiments. First it was shown that 90% of the harvested cells were released from Durapore strips after being shaken in buffer. Second, decreasing numbers of CPMNs were found when performing sequential samplings. Third, when counting CPMNs before and after therapy, a significant decrease in the number of cells was found. Fourth, we could confirm that sites with bleeding on probing did contain a much higher number of CPMNs as compared to nonbleeding sites. Finally, our counting technique confirmed that deeper pockets contain a significantly higher number of CPMNs.

Studies were performed to evaluate the detection of disease-associated bacterial colonization in adult periodontitis patients by the antibody-based Evalusite TestTM (Eastman Kodak Company). The association of test results with disease was assessed by collecting 104 duplicate subgingival plaque samples from 26 patients. Samples were tested for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis and Prevotella intermedia using both microbiological culture and the immunoassay test. The sensitivity and specificity of the 2 methods was calculated using %s of positive results in deep periodontal pockets and negative results in shallow subgingival sites. A cutoff >10(4) cultivable counts yielded the greatest discrimination between health and disease on a cross-sectional basis and established this threshold as clinically relevant for the detection of disease-associated levels of bacterial colonization by these three microorganisms. The clinical detection limit of the immunoassay test was observed to coincide with this threshold of >10(4) cultivable counts. Microbiological testing of the 4 deepest pockets using the immunoassay test was determined to be sufficient to yield a 90% confidence of detecting positive patients in a study with 59 adult subjects. The immunoassay test method was also demonstrated to be effective at detecting bacterial colonization in sets of paper point samples that were pooled for analysis. An overall agreement of 94% (288 of 306) was observed when comparing test results for duplicate sets of pooled and individual samples collected from 51 patients. These studies demonstrate that the Evalusite Test is an effective method for detecting clinically relevant colonization by the test bacteria in patients at risk for periodontal disease.

The present investigation was designed to assess the effects of strips made of different materials on the recovery of enzymes in gingival crevicular fluid (GCF) (Experiment 1), and to examine a possible relationship between lysosomal enzyme activities and number of crevicular polymorphonuclear leukocytes (PMNs) (Experiment 2). In Experiment 1, GCF was collected with capillaries from 14 patients with periodontal disease, and applied on various test strips in microcentrifuged tubes or directly into tubes (controls). Strips were then shaken and centrifuged to elute GCF enzymes. The supernate was used for determinations of alkaline phosphatase (ALP), beta-glucuronidase (BG) and aspartate aminotransferase (AST) activities. The % recoveries were calculated as relative percentages to controls. When using saline as eluent, 90% or more ALP, BG and AST were found to be recovered from strips of durapore and papers. More than 35% of ALP and BG was found to remain on paper points. However, the % recoveries from paper points were improved using eluent with 0.1% (w/v) cetyl pyridinium chloride. In Experiment 2, 54 GCF samples were collected from 3 periodontitis patients by using durapore strips, in order to measure both PMN numbers and BG activities in the same samples. The 2 parameters showed strong and positive correlation with 0.847 (p < 0.001) of the Pearson's correlation coefficient. These findings suggest that durapore is a useful material not only for counting PMNs but also for measuring activities of GCF enzymes. Elevation of BG activities in GCF can be due to increasing numbers of PMNs.

Gingival crevicular fluid (GCF) volume and constituents in static samples were compared to volume and constituents in subsequent GCF samples collected during a 60-min interval. Using deep intracrevicular placement of precut filter paper strips, GCF was collected from interproximal and facial sites from patients with gingivitis (N = 14; 28 interproximal sites, 28 facial sites) and chronic adult periodontitis (N = 11; 26 interproximal sites, 18 facial sites). The strips were inserted for 30 s at 0, 4, 8, 30 and 60 min. The amount of fluid on each strip was determined and microspectrophotometric techniques were used to assess cytoplasmic and lysosomal enzyme activity. Within each group of sites, mean GCF volume showed minimal fluctuation with repeated sampling. In contrast, the static GCF sample contained the greatest amount of total enzyme activity, and differences were detected between groups. The interproximal sites and the gingivitis-facial sites displayed a similar pattern of change in total enzyme activity during the test period. The highest total enzyme activity was observed in the first sample and decreased at 4 and 8 minutes. At 30 and 60 min, the amount of enzyme either remained at the level detected at 8 min, or displayed a mild tendency to recover towards baseline. A different pattern of total enzyme activity was observed for the periodontitis-facial sites, where a significant decrease was first observed at 30 min. Enzyme concentration was higher in the facial sites than the interproximal sites, and enzyme concentration was generally highest in the static samples. The concentration data, however, is difficult to interpret since a number of sites demonstrated a converted GCF volume of 0 microliter. Our data suggests that total enzyme activity and enzyme concentration are generally greater in the static GCF samples compared to subsequent samples.(ABSTRACT TRUNCATED AT 250 WORDS)

A combination of blotting of bacterial colonies on nitrocellulose paper discs and immunoenzymatic detection of bound antigens with specific monoclonal antibodies was used to detect and serotype Actinobacillus actinomycetemcomitans directly off the initial agar culture dish. The A. actinomycetemcomitans antigens, representative of specific colonies, were identified immunoenzymatically using monoclonal antibody specific for a species-specific antigen. The serotype of the bacteria in colonies was identified by dividing the blotting paper into sections and immunoenzymatically identifying the serotype antigens with serotype-specific monoclonal antibodies. This procedure provides for simple, rapid, sensitive and accurate identification and characterization of A. actinomycetemcomitans isolates from the initial isolation agar plates.