Development of a Laser Nephelometric Method for the Quantitation of Human Glycohemoglobins

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Abstract
A nephelometric procedure for quantitative measurement of glycohemoglobin (Hb A1) was developed, evaluated, and compared with the semi-quantitative mini-column chromatographic procedure. Hb A1 was purified from human red cell hemolystate by Bio-Rex 70 ion-exchange liquid chromatography and was used for standards and immunization. The antisera raised in rabbits showed high cross-reactivities with normal human hemoglobin (Hb A). The latter was separated by mixing 25 μl of patients' hemolystate with 2 ml ion-exchange resin suspension for 15 minutes. One hundred microiters of the supernatant was incubated with 900 μl antiserum (dilution 1:50) for 45 minutes at room temperature. Samples were then read on a laser nephelometer. The sensitivity of the assay was found to be 0.1 mg Hb A1. The intraassay relative standard deviation (RSD) was 5.6% and the interassay RSD was 6.5%.