Slot immunoblot assay for detection and quantitation of periodontal disease-associated microorganisms in dental plaque.
A rapid method for qualitative and quantitative detection of specific oral microorganisms from subgingival dental plaque is described. Plaque samples were suspended in phosphate-buffered saline containing protease inhibitors and 0.5% formaldehyde, briefly sonicated to disperse bacterial aggregates, and applied to nitrocellulose membranes in a slot blot manifold. Subsequent incubations with species-specific rabbit antibody and anti-rabbit antibody-alkaline phosphatase conjugate and development with BCIP-NBT substrate resulted in an easily discernible, permanent stain being deposited at the sample application site. Comparison with known concentrations of pure, cultured microorganisms applied to the same membranes permitted qualitative or semiquantitative plaque characterization by visual inspection. Analysis of the blots with a computer-linked flatbed scanner provided quantitative data on microbial content. The reproducibility of the results (standard error of the mean, less than 10%) obtained with slot immunoblotting greatly exceeded that of the results obtained with immunofluorescence analysis (standard error of the mean, greater than 57%). Because it is versatile, rapid, sensitive, reproducible, permanent, and relatively inexpensive, slot immunoblotting lends itself to use in large-scale investigations for the detection and quantitation of specific microbial species.
van Poperin
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Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan, Ann Arbor 49109-0402.
Lopatin
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D000328:Adult; D002241:Carbohydrates / Q000032:analysis; D003773:Dental Plaque / Q000737:chemistry / Q000382:microbiology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting / Q000379:methods* / Q000706:statistics & numerical data; D010510:Periodontal Diseases / Q000382:microbiology*; D016966:Porphyromonas gingivalis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014208:Treponema / Q000302:isolation & purification
D002241:Carbohydrates; D011506:Proteins