The measurement of a glycated protein by immunoassay

• Main Author: Kirkham, Paul A.
• Published: University of Surrey 1989
• Subjects: 572; Blood sugar levels][Diabetes
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328655

The measurement of glycated proteins, in particular, glycated albumin using specific antibodies offers the much-needed possibility of a (semi) automated method for the diagnosis and monitoring of diabetic control. Glycated proteins would appear to be poor immunogens as is evident by the lack of antibodies successfully raised against them when compared against the number of antibodies recognising reduced-glycated proteins. This study has successfully overcome this problem by synthesing several different glycated compounds as haptens and then using them in conjunction with a carrier protein to raise antisera in sheep. The binding of one of these antisera, with a titre 1:100000 showed considerable displacement when incubated with diabetic plasma at various dilutions. Western blot analysis on human plasma confirmed that the antiserum specifically recognised a continuous epitope on glycated human serum albumin. Affinity purified antibodies were used to develop both an indirect competitive ELISA and later a direct non-competitive ELISA for glycated serum albumin which does not require prior reduction of the glycated protein to the glucitol form. These assays have a dynamic range at 0 to 100 mug/ml and 0 to 50mug/ml of glycated human serum albumin respectively. The competitive ELISA exhibited < 0.15% cross reactivity with both sodium borohydride and sodium periodate treated human serum albumin. Further work was needed to be undertaken to develop a rugged ELISA that could discriminate between diabetics and normals by routinely measuring glycated human serum albumin levels.