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A sensitive method has been developed using 125I-labelled anti-IgG which allows detection of IgG present on the red cell surface. By suitable absorption and correction for non-specific binding to trypsinized red cells, a reproducible, accurate method of quantitating IgG absorbed onto red cells is obtained. The number of IgG molecules present on normal cells was found to range from 5 to 90 with an average of 39. Studies of correlation of agglutination in the antiglobulin test with numbers of IgG molecules on the cell were also undertaken.

The number of IgG molecules bound to the erythrocyte surface for a given agglutination score in the antiglobulin test was studied with several different examples of anti-D, anti-E, anti-c, anti-Kell, anti-Fya, anti-Jka and immune anti-A antisera. The serological scores show a significant correlation with the mean values for bound IgG molecules within a restricted range, although the number bound for a given score may vary by up to 20%. The limit of detection was 100-120 IgG molecules per cell and when over 1,000 were bound, the cells were completely agglutinated. Anti-Kell bound under low ionic strength saline conditions required a greater number of molecules for a given agglutination strength. The relatively low levels of bound IgG necessary to give strong agglutination make the direct antiglobulin test (DAT) less valuable for following the progress of auto-immune haemolytic anaemia (AIHA) than a quantitative test. The latter test does not, however, provide any additional information in AIHA cases with a negative DAT as in these the anaemia does not appear to be due simply to the number of bound IgG molecules. Detection of certain antibodies may not be achieved simply by increasing the sensitivity of the antiglobulin test when correctly performed.

Investigations into the IgG sub-types and number of molecules of IgG present on the red cells of 22 apparently normal healthy blood donors with positive direct antiglobulin tests are described. In all cases, the sub-type was IgG1 or IgG4, and none had more than 1000 mol of IgG per red cell. It is suggested that sequestration of IgG-coated cells only occurs when the number of IgG1 mol per cell reaches a certain level.