Development of an Immunoassay for the Detection of Minute Amounts of IgG‐Coated Erythrocytes in Whole Blood and Its Application for the Assessment of Fc‐Mediated Clearance of Anti‐D‐Coated Erythrocytes in vivo

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Abstract
A rapid and sensitive immunoassay for the detection of minute quantities of IgG‐coated erythrocytes in whole blood was developed. Washed red blood cells were incubated in two steps with anti‐human IgG antiserum followed by 125I‐labelled protein A. The assay was able to detect amounts of sensitized erythrocytes as small as 0.5 ml of packed erythrocytes in a total blood volume of 5 liters and hematocrit 40%. A linear relation between increasing amounts of IgG‐coated red cells in whole blood and the binding of 125I‐labelled protein A was obtained. We applied the technique on the assessment of the removal of IgG anti‐D‐coated erythrocytes from the circulation of test individuals. T1/2 for the elimination of approximately 4 ml packed red cells sensitized with 62 μg of anti‐D in 14 normal subjects was 20±5 min (mean±SEM). A splenectomized person did not clear the injected cells from the circulation during the test period of 70 min. If a standard curve was constructed the total blood volume in the test subjects could be calculated. This value correlated well (r = 0.99) with the blood volume calculated from the height and weight of the test individuals.