A Quantitative Assay for Subclassing IgG Alloantibodies Implicated in Hemolytic Disease of the Newborn

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Abstract
Traditionally, IgG subclassing has been performed using qualitative assays. Quantitation of IgG subclasses may have prognostic value in evaluating alloimmunized pregnancies. A quantitative enzyme-linked immunosorbent assay (ELISA) was implemented for measuring IgG subclasses of red blood cell (RBC) antibodies (AB) isolated by adsorption/elution from the sera of alloimmunized pregnant women. The assay is a sandwich enzyme immunoassay using monoclonal antibodies specific for the relevant IgG subclasses and anti-human IgG peroxidase conjugate to quantitate the amount of bound IgG. The sensitivities of the assay for IgG1, 2, 3, and 4, respectively, were 4, 23, 4 and 2 micrograms/l. The results for each subclass for a given AB were expressed as a percentage of the total. In a series of pregnant mothers with ABs: E (4), Fya (2), Jka (1) and S (1), the mean percentage +/- 1SD of each subclass was: IgG1 61 +/- 34; IgG2 14 +/- 22; IgG3 18 +/- 28 and IgG4 4 +/- 17. IgG1 or IgG3 accounted for greater than 50% of the AB subclass distribution in 5 cases that resulted in hemolytic disease of the newborn (HDN). Although only a small number of samples was studied, changes in the concentrations of IgG1 or IgG3 during gestation suggest a correlation with the presence or absence of HDN. The ELISA may be used to quantitate the IgG subclasses of RBC ABs and may be valuable in predicting the severity of HDN in alloimmunized pregnancies.