Development of a Rapid Diagnostic Test for the detection of Streptococcus mutans in Oral Fluids for the Diagnosis of Dental Caries
Date
2017-12
Authors
Braddock, Kelly
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Abstract
The personal and financial impact of common and often overlooked oral diseases, such as dental caries, is sizable and prevalent throughout the world. Rapid diagnostic tools continue to be developed to prevent, diagnose and monitor treatment responses of a variety of diseases including, dental caries. This assay, like many other point-of-care diagnostics, is based on the lateral flow immunoassay design. Initial development required identification of two antibodies that specifically recognize antigens on the surface of Streptococcus mutans, and are compatible as a matched pair. To identify a matched pair, antibodies were first purified from hybridoma cell culture supernatant, by affinity chromatography, and screened against two S. mutans strains of differing serotypes. The two hybridoma cell lines for which antibody was successfully purified, were screened and both indicated activity against one S. mutans strain of the most common serotype, serotype c. However, only one of the purified antibodies was able to recognize the non-serotype c strain. Further assay development focused only on the strain of S. mutans recognized by both antibodies. Conjugation to biotin allows the detection antibody to form a complex with the reporter molecule HRP, which is conjugated to streptavidin. A portion of both antibody stocks were conjugated to biotin and each of them was tested in both the detecting and capture positions, in order to determine the optimal orientation of the matched pair. Only one orientation recognized S. mutans within the lateral flow assay. Initial experiments indicated that the lower limit for S. mutans detection was within the clinically relevant range, for which concentration of salivary S. mutans can be used to diagnose dental caries. After identifying a matched pair, detergent concentration as well as blocking conditions were optimized. To assess analytical performance within a realistic matrix, a dose response curve was created from samples made in human pooled saliva. The dose response indicated results of the assay at varying concentrations were reproducible, but detection of S. mutans occurred only at concentrations much higher than in previous testing. This discrepancy is likely due to matrix effects within saliva, including presence of endogenous proteins and high viscosity. Future testing to optimize the assay within saliva, may improve its detection capabilities by mitigating some of these matrix effects, through addition of detergents or mucolytic agents.
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Keywords
Lateral flow, Dental caries
Citation
Braddock, K. (2017). <i>Development of a rapid diagnostic test for the detection of Streptococcus mutans in oral fluids for the diagnosis of dental caries</i> (Unpublished thesis). Texas State University, San Marcos, Texas.