Monoclonal RhD antibodies (anti-RhD) may potentially be used in preventing Rh alloimmunisation of women delivering RhD-positive babies, and thereby preventing haemolytic disease of the newborn (HDN). Binding and molecular analyses were performed on a range of monoclonal anti-RhDs to understand the interaction between the antibody and antigen. A flow cytometry (FCM) assay system was developed to analyse the binding of the anti-RhD to human group O R<sub>1</sub>R<sub>2</sub> red blood cells (RBC). A panel of 30 anti-RhD preparations were studied using this assay and Vmax (maximum binding) and KD (equilibrium constant) were determined for each antibody. The antibodies were categorised into 3 groups (low, medium and high binders) according to their Vmax. Furthermore, the Vmax was converted to the number of antibody molecules bound per RBC (NMBR) by using a correlation curve generated from running parallel FCM and radioiodination assays (RIA). Scatchard analysis of the RIA data indicated that the total NMBR for O R<sub>1</sub>R<sub>2</sub> RBC was 27,300 antigen sites/cell. Molecular analysis involved cloning and sequencing of 11 anti-RhD. Heavy chains (HC) preferentially used gene segments from the VH3 and VH4 families, and kappa chain (κ LC) usage was restricted to DPK9. Four sets of antibodies, showed restricted D gene segments (encode part of the HCDR3) which indicated possible importance for epitope specificity. Analysis of V gene sequence indicated that common VH and VL pairings were found used by the medium binders. The high and low binders had unique VH and VL pairings, although the high binders also showed greater somatic mutations from their respective germline genes. It was concluded that the fit of the antibody to the RhD antigen is dependent on both the VH and VL usage and pairing, and that the precise epitope specificity of these antibodies may require HCDR3 interaction.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:342706 |
| Date | January 1999 |
| Creators | Perera, W. Shermal |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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