TIME-RESOLVED FLUORESCENCE IMAGING IN ISLET-CELL AUTOANTIBODY QUANTITATION

The prodromal period of insulin-dependent diabetes mellitus (IDDM) is characterized by circulating islet cell autoantibodies (ICA) and other beta cell specific autoantibodies. Despite biochemical characterizati on of the major beta cell autoantigens insulin, glutamic acid decarbox ylase and protein tyrosine phosphatase and development of the respecti ve antibody assays, ICA has remained the standard in IDDM prediction. Conventional ICA quantitation using classic fluorochromes is prone to errors since fluorescence intensity is estimated subjectively using th e human eye, which is also unable to differentiate specific signals fr om non-specific signals and autofluorescence. Using Eu3+-chelate label led anti-human polyclonal IgG (decay time 1000 mu s) as the secondary antibody in time-resolved fluorescence imaging (TRFI), the chelate and autofluorescence signals (typical decay time < 100 ns) are fully sepa rated. The image is recorded using an optically gated cooled digital C CD camera. The specificity of the ICA signal is further improved by in teractive analysis of the image. Signal detection is objective, the si gnal-to-background ratio improves, and ICA quantitation is possible us ing undiluted serum. Of 57 consecutive new-onset IDDM patients, 55 (96 .5%) were ICA positive in the new assay while 51 (89.5%) were positive in the conventional assay suggesting that the sensitivity of TRFI exc eeds that of the IAA, GAD(65) and IA-2 autoantibody assays combined. F or later comparisons, the stained slides may be stored in the light fo r years without any decrease in specific fluorescence. (C) 1997 Elsevi er Science B.V.