Quenching of fluorescence by glycated haemoglobin: early development of a novel assay

A wide range of methods have been adopted for the determination of glycated haemoglobin (either HbA(1c) HbA1 or total glycated haemoglobin GHb) and have been reviewed recently. Methods relying on the change in charge from glycation (Ion-exchange chromatography and electroendosmosis) and methods relying on a change in structure (immunoassay) are susceptible to interference from abnormal haemoglobins. Affinity methods, utilizing the binding of boronic acids to the cis-diol group on glucose, do not suffer from these interferences and so are suitable for populations with a high incidence of abnormal haemoglobins. Current affinity methods (manual mini columns, automated high-performance liquid chromatography and ion-capture affinity) all involve a separation step. We present here the preliminary results from the development of a novel, fluorescent, non-separation affinity method for the determination of total glycated haemoglobin. The principle of the method is that the emission of certain fluorescent derivatives of boronic acids is quenched when bound to glycated haemoglobin but not when unbound.