Development of an oral fluid assay for the detection of uncontrolled diabetics using glycated albumin as a marker

The purpose of this project was to identify a diabetic marker in oral fluid and to use the marker to develop an assay sensitive enough to distinguish between non-diabetics or controlled diabetics and uncontrolled diabetics. The objective was to design an oral fluid enzyme linked immunosorbant assay (ELISA) capable of accurately and quickly identifying undiagnosed diabetics. Such an assay could replace more expensive and intrusive methods of diabetic diagnosis currently being used, such as hemoglobin A1c (HbA1c) measurement. We examined two possible oral fluid diabetic markers: advanced glycated end products (AGE) complexes consisting of albumin and various oligosugars, and glycated albumin (GA).; The project involved the development of two separate immunoassays: the first to determine the amount of marker (GA or AGE) in a collected oral fluid sample, and the second to determine the amount of total albumin in the sample. The ratio of marker verses total albumin is then used to obtain the percent of AGE or GA in the sample. The percent marker is subsequently employed to determine if the sample is from a diabetic individual. In normal (non-diabetic) individuals, 1–3% of albumin is glycated but that percentage rises to 3–6% in diabetics.; Monoclonal and polyclonal antibodies produced to detect AGE and GA in direct and indirect assay formats were found to have cross-reactivity with non-albumin glycated proteins. This cross reactivity caused problems in overall assay accuracy and sensitivity. The presence of the cross reactivity coupled with the presumption that AGE in oral fluid are unlikely to occur at measurable levels led to the development of a two-step assay format for the quantitation of GA rather then AGE. The two-step assay format employed column separation of glycated albumin from non-glycated albumin followed by glycated and non-glycated albumin quantification using the total albumin ELISA developed for the project. This two-step process successfully eliminated albumin cross reactivity problems encountered in other assay formats.