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

Ann Clin Biochem 2000;37:372-379
doi:10.1258/0004563001899302
© 2000 Association for Clinical Biochemistry

 

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Original Articles


Stuart Blincko,
D Avid Colbert,
W Garry John and
Raymond Edwards

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% differencefrom 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 methodbut with significantly less susceptibility to interferences.As such the method should be a useful aid in the managementof diabetes.


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