Quantitation of red cell-associated IgG using an immunoradiometric assay

In this report, we describe a sensitive immunoradiometric assay (IRMA) for quantitating IgG on the surface of red cells. Washed red cells were prepared to a purity of greater than 99.9 percent. Varying dilutions of these cells were incubated with a fixed concentration of 125I-anti-IgG. After equilibrium was achieved, the unbound 125I-anti-IgG was measured by the addition of IgG covalently linked to agarose beads. The red cells were lysed by detergent, and the 125I-anti-IgG bound to the IgG- beads was measured. The amount of IgG on the red cells was determined by relating the concentration of test red cells causing 50 percent inhibition of binding of the 125I-anti-IgG to the IgG-beads to 50 percent inhibition of binding caused by the IgG standard. Using this assay, the red cell-associated IgG (RCA-IgG) of 20 healthy male and female controls with normal hemoglobin concentrations was 7.23 +/− 6.11 fg IgG per 10(3) cells (mean +/− 2 SD). The mean RCA-IgG on washed cells from 34 different tests performed on 19 anemic patients with clinically diagnosed autoimmune hemolytic anemia was 176.1 +/− 375.6 fg IgG per 10(3) cells. There was no correlation between the levels of RCA- IgG and the hemoglobin levels or reticulocyte counts in these patients.