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.

Quantitation of red cell‐associated IgG using an immunoradiometric assay Journal Articles