Diagnosis of militarily relevant diseases using oral fluid and saliva antibodies: Fluorescence polarization immunoassay

Military Medicine,  Nov 2003  by Cullum, Malford E,  Lininger, Linda A,  Schade, Sylvia Z,  Cope, Stanton E,  Et al

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This laboratory is developing fluorescence polarization (FP) methods as diagnostic tools to assay antibodies in saliva and other oral fluids. FP provides quantitation of molecular interaction, such as antigen-antibody binding, of a single, smallvolume sample in real time and without prior separation of components such as blood cells. There is potential for widespread use of these homogeneous assays as noninvasive tests, especially as more compact, simplified fluorescence polarimeters become available. FP tests can be designed that are applicable to a wide spectrum of microorganisms and may be used in a clinic or far-forward deployed setting to aid in diagnosis of disease or verification of vaccination. Rapid salivary diagnostics, including FP, have been identified by the Office of Naval Research as requirements for future naval capabilities in basic and applied medical research for warfighter protection in casualty prevention. The applications of FP salivary diagnostics for determination of tuberculosis exposure and of anthrax immunization status are discussed as examples.

Introduction

Oral fluids have been increasingly recognized as acceptable alternatives to serum for use in diagnostic tests for certain hormones, drugs, antibodies, and antigens. However, diagnostic assays using saliva and other oral fluids appear as entries in the National Library of Medicine MEDLARS database with a frequency of only 1 in 30 and 1 in 100, respectively, when compared with entries in which blood serum is reported. Oral fluids are collected without pain, needle sticks, or religious and social prohibitions, and use of these involves minimal risk or exempt protocols for the use of human subjects. The Department of Defense has made salivary diagnostics a future naval capability, and both the Office of Naval Research and the Military Infectious Disease Research Program of the U.S. Army have funded projects to investigate the use of oral fluids or saliva to diagnose disease or monitor immunization status. The military has had a long-standing interest in saliva with Office of Naval Research, compiling in 1960 a review of all saliva references in the literature from 1888 to 1957.1 In 1965, the U.S. Army Medical Research Development Command supported a contract that published one of the first reports of human salivary antibodies to indigenous bacteria.2 The U.S. Department of Agriculture recently sponsored a symposium to advance noninvasive or minimally invasive technologies to monitor health and nutritional status in the Special Supplemental Nutrition Program for Women, Infants, and Children using saliva as a diagnostic tool.3,4 These considerations and continuing improvements in standardization of collection methods make these body fluids the diagnostic media of choice in the 21st century.

Recent reviews of the clinical chemistry and microbiology of saliva5 and the roles of saliva in health and disease,6 in diagnosing periodontal disease,7 and as a diagnostic fluid8 indicate the growing prominence saliva plays in medical diagnostics.9,10 Only recently has "oral fluid" been defined, perhaps, as a better alternative (see below). Saliva6 and "oral fluid"11 are distinct biochemically. They generally reflect the serum pool, but neither is purely a passive ultrafiltrate of serum as previously thought.12 The presence of mucins, the polyanionic glycoproteins that increase salivary viscosity, and oral flora have been largely responsible for the lack of popularity of saliva in clinical research. Saliva presents several challenges for diagnostics. Limited reference data have been published for saliva.13

Standardized saliva14 and "oral fluid"15 collection devices have now become available and should contribute to further investigations. Gingival crevicular fluid and oral mucosal transudate arise due to hydrostatic pressure of the capillaries and venules associated with the lingual or buccal epithelium. They may offer less variation than saliva and a better alternative to serum for antibody detection.16 Gingival crevicular fluid is similar to serum in protein composition but is significantly lower in protein concentration, being approximately 3% of the protein levels in blood.17,18 Gingival crevicular fluid volume is approximately 1% of total saliva volume in the healthy mouth19 and is obtained by inserting an absorbent paper into the pocket or sulcus of a tooth (between the tooth and gingiva) after clearing the supragingival plaque.20 Its medical and dental use has not been reviewed since the 1970s.21,22 Oral mucosal transudate is collected by placing a thick pad against the buccal mucosal surface juxtaposed between the parotid duct and the gingival crest.11 A current oral mucosal transudate device uses a salt-impregnated pad that is subsequently treated to release the antibody-containing fluid and retain the glycoproteins on the pad, yielding "oral fluid." This "oral fluid" may contain three to four times the concentration of IgG as that found in saliva depending on the investigator.11,23 One such "oral fluid" collection device has been licensed by Abbott Laboratories and eliminates the necessity for venipuncture.24