Measurement of plasma glycoalbumin levels with a monoclonal antibody based ELISA

Abstract

We have reported the establishment of hybridomas secreting monoclonal antibody that specifically recognizes glycated albumin. The antibody, raised in mice immunized with nonenzymatically glycated albumin isolated from human plasma, recognizes glycated epitopes residing in albumin but not in other plasma proteins, and does not react with unglycated albumin. We now report utilization of this antibody to measure the amount of nonenzymatically glycated albumin in human plasma. When immobilized onto microtiter wells in ELISA format, A717 yields a linear dose-response relationship upon incubation with authentic purified glycoalbumin. This allows construction of standard curves from which the amount of circulating glycoalbumin can be determined. The glycoalbumin level (mean ± SEM) in 12 samples from nondiabetic subjects was 2.4 ± 0.22% of total albumin. In normal plasma spiked with known amounts of purified glycoalbumin, values measured with this assay were close to 100% of expected values. Glycoalbumin levels in samples from 25 diabetic subjects ranged from 1.6 to 11.6%, with a mean ± SEM of 4.5 ± 1.2%. Glycoalbumin levels in diabetic samples correlated significantly ( r = 0.93) with glycohemoglobin values. The ability of this assay to quantitate glycoalbumin, an index of the prevailing blood glucose concentrations over the preceding 2–3 weeks, makes it a favourable candidate for utilization in the clinical setting to monitor glycemic control in diabetic subjects.