Use of the DAF Assay to Assess the Functional Properties of Polyclonal and Monoclonal Rh D Antibodies

Abstract

The mechanism whereby passive Rh (D) immunoglobulins suppress the fetomaternal alloimmunization is still unclear. New in vitro tests are needed to better characterize the functional properties of polyclonal anti-Ds. The DAF assay was developed to monitor the antibody-dependent cell-mediated cytotoxicity (ADCC) and the phagocytosis of anti-Rh (D)-sensitized RBCs by effector cells. The principle of this test is based on the oxydization of the 2,7-diaminofluorene (DAF) by the pseudoperoxidase activity of free hemoglobin. The reaction is proportional to the hemoglobin concentration. This test was performed to determine and emphasize the efficacy of different polyclonal anti-D immunoglobulin preparations to mediate lysis and phagocytosis of sensitized RBCs by human peripheral mononuclear cells. The functional properties of different human RhD monoclonal antibodies were also analyzed and compared. The test was found to be convenient to perform and allowed the avoidance of radioactive labelling of RBCs for ADCC studies. It is mainly useful for the direct quantitation of phagocytosis.

Flow–Cytometric Method for the Quantitation of the Fc Function of Intravenous Immunoglobulin Preparations

“…These assays of Fc function either measure rosette formation or lytic activity caused by the antibody–dependent cellular toxicity (ADCC) of lymphocytes or monocytes against Rh–D–positive cells. Most ADCC assays, require radioactive labelling of red blood cells [11]though technical variations of this assay detect haemolysis by the pseudoperoxidase activity of the free haemoglobin [12]. All such assays are subject to interlaboratory variability as assay conditions are difficult to standardise.…”