Evaluation by enzyme-linked immunosorbent assay of IgG anti-D and IgG subclass concentrations in immunoglobulin preparations

BACKGROUND: Anti-D immunoglobulin preparations are injected to prevent hemolytic disease of the newborn. The concentration of IgG anti-D in these preparations is usually determined by an automated hemagglutination technique using as a reference a calibrated preparation of anti-D but the method requires special equipment and cannot be routinely applied to measure the IgG subclasses of anti-D in these preparations. STUDY DESIGN AND METHODS: Taking advantage of a recently described enzyme-linked immunosorbent assay (ELISA) for the determination of the anti-D concentration in sera of alloimmunized pregnant women, IgG anti-D and IgG subclass concentrations were measured in the international reference preparation (IRP) coded 68/419, 10 anti-D immunoglobulin preparations, and sera of 15 D-immunized volunteers. RESULTS: An IgG anti-D concentration of 61.5 +/- 4.8 mu g per ampoule (mean +/- SD) was found by ELISA in IRP 68/ 419. This result was in agreement with previous determinations obtained by radioimmunoassay (60 mu g/ampoule). The IgG subclass concentration of anti-D in this preparation was 48.4 mu g of IgG1 (78.6%), 3.0 mu g of IgG2 (4.8%), 9.7 mu g of IgG3 (15.8%), and 0.4 mu g of IgG4 (0.7%). The mean proportion of IgG subclasses of anti-D in 10 immunoglobulin preparations was similar (81.7% for IgG1, 5.0% for IgG2, 12.7% for IgG3, and 0.6% for IgG4). In the sera of 15 immunized volunteers, the IgG anti-D concentration varied from 3.1 to 68.4 mu g per mt. The mean IgG subclass composition of anti-D was 79.3 percent for IgG1, 2.2 percent for IgG2, 18.1 percent for IgG3, and 0.4 percent for IgG4. The proportions of IgG3 anti-D in these sera were found to range between 1 percent and 87 percent, as in the sera of D-alloimmunized pregnant women. CONCLUSION: ELISA provides an alternative to the radioimmunoassay and the automated hemagglutination technique. In addition, it allows the evaluation of the absolute concentration of each IgG subclass of anti-D in immunoglobulin preparations and necessitates only the conventional equipment required for an immunoenzymatic assay.