An enzyme‐linked immunosorbent assay for the quantitation of IgG anti‐D and IgG subclasses in the sera of alloimmunized patients

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Abstract
BACKGROUND: IgG subclass composition of maternal alloantibodies to the D antigen seems to play a role in the severity of hemolytic disease of the newborn. The subclassing of IgG anti‐D is usually performed by hemagglutination techniques, but the results are not quantitative and sometimes are difficult to interpret. Thus, there is a need for quantitative methods. STUDY DESIGN AND METHODS: The aim of this study was to develop an enzyme‐linked immunosorbent assay (ELISA) for the quantitation of specific IgG anti‐D and IgG subclasses in the sera of alloimmunized patients. Group O R1R2 red cells were sensitized with anti‐D. Red cell membranes were solubilized with nonionic detergent. IgG and IgG subclasses were measured by a sensitive and reproducible immunocapture ELISA. A serum calibrated for its IgG subclass content was used as a reference, and the anti‐D preparation 68/419 was used as an internal control. Optimal conditions for the detection of IgG anti‐D and IgG subclasses by ELISA were studied. The absolute concentration and the proportions of IgG subclasses were determined in the sera of 14 pregnant women. RESULTS: A close parallelism was observed between dilutions of the IgG reference serum and the IgG anti‐D solubilized from sensitized RBCs. The sum of IgG anti‐D subclass concentrations, determined by the ELISA, correlated well with other quantitative methods. CONCLUSION: The method described is sensitive and can be used routinely for the quantitative determination of specific IgG anti‐D and IgG subclasses in sera.