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

A Simple non-separation assay For the measurement of total glycated serum p rotein is described. It was found that the fluorescence intensity of a solu tion 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 whic h 10 mu L of serum is added to 4 mL of a solution of the fluorescein-boroni c 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 les s than 2.1% (concentration range 190-660 mu mol/L); inter-assay precision f or seven samples in 10 assays was less than 2.5% (over the same concentrati on range). Dilution of serum that had a high concentration of total glycate d protein showed the assay to be linear. Serum samples (with low, medium an d high total glycated protein concentrations) showed less than 2.1% differe nce from base results with added glucose (up to 60 mmol/L), less than 9.7% difference with added bilirubin (up to 250 mu mol/L) and less than 6.9% wit h added triglycerides (up ro 50 mmol/L). Addition of haemoglobin (up to 0.9 g/dL) with high glycation (11.7% HbA(1c)) to plasma (298 mu mol/L total gl ycated protein) showed less than 10% difference from the base result. Assay s performed over a range of temperatures (12-14 degrees C) showed no signif icant differences in the results. The assay gives similar results to the cu rrently used NTB method but with significantly less susceptibility to inter ferences. As such the method should be a useful aid in the management of di abetes.