A reliable non-separation fluorescence quenching assay for total glycated serum protein: a simple alternative to nitroblue tetrazolium reduction

Abstract

A simple non-separation assay for the measurement of total glycated serum protein is described. It was found that the fluorescence intensity of a solution of a fluorescein-boronic acid derivative was quenched in proportion to the amount of serum added. This led to the development of an assay in which 10 µL of serum is added to 4 mL of a solution of the fluorescein-boronic acid derivative and the fluorescence intensity is measured after 15 min. The results, as measured by drop in fluorescence intensity, calibrated by a single standard, were compared with the results for nitroblue tetrazolium (NBT) reduction of fructosamine and showed good correlation ( r=0·936, n=114). The intra-assay precision (seven samples each measured 10 times) was less than 2·1% (concentration range 190-660 µmol/L); inter-assay precision for seven samples in 10 assays was less than 2·5% (over the same concentration range). Dilution of serum that had a high concentration of total glycated protein showed the assay to be linear. Serum samples (with low, medium and high total glycated protein concentrations) showed less than 2·1% difference from base results with added glucose (up to 60 mmol/L), less than 9·7% difference with added bilirubin (up to 250 µmol/L) and less than 6·9% with added triglycerides (up to 50 mmol/L). Addition of haemoglobin (up to 0·9 g/dL) with high glycation (11·7% HbA 1c) to plasma (298 µmol/L total glycated protein) showed less than 10% difference from the base result. Assays performed over a range of temperatures (12- 34 C) showed no significant differences in the results. The assay gives similar results to the currently used NTB method but with significantly less susceptibility to interferences. As such the method should be a useful aid in the management of diabetes.