A competitive enzyme-linked immunoassay using erythrocytes fixed to microtitre plates for anti-D quantitation in...

The batch control of anti-D immunoglobulin for prevention of haemolytic disease of the newborn necessitates assessment of its potency. Anti-D quantitation is usually performed using automated haemagglutination methodology although this can only be carried out in specialist centres. The aim of this study was to develop a simple and robust assay for anti-D quantitation.

We developed a competitive enzyme-linked immunoassay (EIA) in which unlabelled anti-D immunoglobulin and a biotinylated monoclonal anti-D compete for red cell binding. Binding of biotinylated anti-D is detected using an alkaline-phosphatase-labelled avidin preparation. The assay is conveniently carried out using erythrocytes fixed to microtitre plates.

The competitive EIA was specific for anti-D activity, highly reproducible and showed good correlation with manufacturers' potency estimates using automated haemagglutination. The assay was quick and simple to perform using freshly prepared or stored plates, and the biotinylated monoclonal anti-D could be lyophilized in ampoules for distribution as a standardized reagent.

The competitive EIA described can be used for the specific quantitation of anti-D and provides a robust alternative method to automated haemagglutination.