Detection of erythrocyte membrane components in hemoglobin-based blood substitutes

Two methods for the detection of membrane components in human stroma-free hemoglobin solutions are described. The first is a phospholipid assay with a detection limit of 0.5-1 nmol phospholipid/ml hemoglobin-solution. For the detection of membrane proteins an immunoassay with a monoclonal antibody against glycophorin alpha was developed (detection limit 0.01% of the original amount). These methods were used to determine the purity of Hb solutions prepared in two different ways. Hb solutions prepared by filtration of red blood cells, gradually swollen in hypotonic buffer, contained 0.25% of the original amount of phospholipid and no detectable glycophorin alpha. For Hb solutions prepared in a similar way from red blood cells lysed in water, the values for phospholipid and glycophorin alpha were 2.5% and 0.06%, respectively. The determination of both glycophorin alpha and phospholipid gives a useful indication of the purity of Hb solutions.