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 characterization of the major beta cell autoantigens insulin, glutamic acid decarboxylase and protein tyrosine phosphatase and development of the respective 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 the human eye, which is also unable to differentiate specific signals from non-specific signals and autofluorescence. Using Eu(3+)-chelate labelled anti-human polyclonal IgG (decay time 1000 microseconds) as the secondary antibody in time-resolved fluorescence imaging (TRFI), the chelate and autofluorescence signals (typical decay time < 100 ns) are fully separated. The image is recorded using an optically gated cooled digital CCD camera. The specificity of the ICA signal is further improved by interactive analysis of the image. Signal detection is objective, the signal-to-background ratio improves, and ICA quantitation is possible using 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 exceeds that of the IAA, GAD65 and IA-2 autoantibody assays combined. For later comparisons, the stained slides may be stored in the light for years without any decrease in specific fluorescence.