QUANTITATION OF ERYTHROCYTE-BOUND IGG SUBCLASS AUTOANTIBODIES IN MURINE AUTOIMMUNE HEMOLYTIC-ANEMIA

A quantitative and sensitive cellular enzyme-linked immunosorbent assa y was developed for determining the number of molecules of IgG of each subclass bound to the surface of murine red blood cells (RBC). To dev elop standard titration curves, RBC from normal mice were treated with tannic acid and coated with a known concentration of purified myeloma of each Ige subclass. The quantity of each subclass bound to the surf ace of erythrocytes was determined by calculating the protein concentr ation of the bound IgG, which was then converted into number of molecu les of IgG/RBC. The assay was used to quantify the number of autoantib odies of all four IgG subclass bound to the erythrocytes of mice injec ted with rat RBC. Twenty one days after the first immunisation, a mean number of 84,000 molecules of IgG1/RBC were detected, which increased to 114,500 molecules/RBC on day 28. On days 55 and 96 the mean concen tration of IgG1 remained high, however by day 110 the mean level of Ig G1 had decreased slighty to 69,500 molecules/RBC. By contrast, the mea n concentration of 1gG2a autoantibodies was considerably lower through out the experiment, starting at 40,200 molecules/RBC on day 21 and dro pping to 2,500 molecules/RBC by day 110. The mean quantities of IgG2b and IgG3 autoantibodies were similar to each other, and intermediate b etween the levels of IgG1 and IgG2a autoantibodies.