An enzyme-linked direct antiglobulin test for assessing erythrocyte bound immunoglobulins

An enzyme-linked direct antiglobulin test (DAGT) for assessing erythrocyte-bound IgG, IgM and IgA is described. The test is carried out in microtitre plates using heavy chain-specific, alkaline phosphatase-linked, goat anti-human globulin reagents with p-nitrophenyl phosphate as substrate. Results are expressed in optical density (OD) units per 3.6 X 10(7) red cells. The method is reproducible, with coefficients of variation of 0.056, 0.093 and 0.087 for IgG, IgM and IgA respectively. The linear relationship between the amount of red cell-bound antibody and the OD reading for each immunoglobulin class shows that the method is suitable for quantitative studies. Healthy individuals were found to have small amounts of immunoglobulin bound to their red cells with mean values of 0.251, 0.087 and 0.128 OD units per 3.6 X 10(7) red cells for IgG, IgM and IgA respectively; there was no difference between male and female subjects. In the clinical situation, the enzyme-linked DAGT was considered to show significantly increased amounts of cell-bound immunoglobulin when the results were more than three standard deviations above the mean and the quantitative results permitted an accurate assessment of the progress and response to treatment of patients with autoimmune haemolysis.