Assay of Human Anti-D Immunoglobulin (Case Study)

Anti-D immunoglobulin is used for the prophylaxis of the Rh disease (also Rhesus disease, haemolytic disease of the newborn (HDNB) or Morbus haemolyticus neonatorum). Rh disease occurs when a Rh negative mother has given birth to a Rh positive baby and subsequently becomes pregnant with another Rh positive child. During the first pregnancy and the act of birth a minimal amount of the baby's blood enters the mother's body. Since she is Rh negative, her body immediately produces antibodies (including IgG) against the Rhesus antigens on her baby's erythrocytes. During the second pregnancy the IgG is able to pass through the placenta into the embryo, where it leads to agglutination and destruction of erythrocytes. The common way to prevent this harmful disease is to vaccinate the mother immediately after the birth of her first child: she is treated with anti-D antibodies, so that the foreign erythrocytes are destroyed before her immune system can discover them.

The potency of human anti-D immunoglobulin may be determined by the following methods:

  • Comparison of the quantity that is necessary to produce agglutination of D-positive red blood cells with the quantity of a reference preparation.

  • Determination of the potency by a competitive ELISA on erythrocyte-coated microtitre plates. This method is based on the competitive binding between a polyclonal anti-D immunoglobulin preparation and a biotinylated monoclonal anti-D antibody directed against a D-antigen specific epitope.

  • Determination of the potency by a flow cytometric method. This method is based on the specific binding between anti-D immunoglobulin and D-positve red blood cells.

Each of the methods listed above should be examined with the usual statistical models given in chapter <1034> of the US Pharmacopeia or chapter 5.3 of the European Pharmacopoeia. Therefore the linear range of the dose response curve is used to estimate the potency of the test preparation in comparison to the reference preparation. As you will see, PLA 3.0 is a very helpful tool for this task.

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