Monoclonal antibodies as tools in antigen detection assay and vaccine development : design of a sensitive detection test for Brucella bacteria and profiling of the malaria vaccine candidate antigen reticulocyte-binding homolog 2 (PfRH2)

We aimed at identifying immunodominant Brucella antigens for implementation in new detection tools or for subunit vaccine development. In particular, our strategy was to produce Brucella cell surface antigen-specific monoclonal antibodies (mAbs) for the development of an antigen capture assay for the detection of Brucella cells as potential bio threat agents in complex samples. We generated a panel of Brucella lipopolysaccharide (LPS)-specific mAbs by immunising mice with inactivated B. melitensis and B. abortus cells. The mAbs recognised Brucella species with ‘smooth’ LPS independently of the way how the bacterial cells were inactivated. Two mAbs were implemented into a bead-based Luminex assay detecting ‘smooth’ Brucella spp. with species-dependent detection limits of 2 x 102 to 8 x 104 cells per mL. Integration of the Luminex assay into a multiplex format enabled simultaneous detection of Brucella spp. and three other bio threat agents within a single sample. The developed Luminex assay may be applied for the detection of whole Brucella cells both in natural Brucella outbreak and in bioterrorism attack scenarios.
We also tried to generate mAbs against Brucella cell surface proteins from mice immunised with inactivated whole Brucella cells. While serum antibody responses against both LPS and protein antigens were seen in Western blotting analyses, attempts to generate protein-specific mAbs failed, most likely due to the immunodominant nature of the LPS. Western blot analyses with Brucella lysate also identified antibodies against some immunodominant Brucella proteins in the serum of cattle naturally infected with Brucella spp., however, identification of the recognised proteins with a Brucella-specific peptide microarray failed.
In a second part of the thesis we aimed at evaluating the potential of the Plasmodium falciparum reticulocyte-binding homolog 2 (PfRH2) present in the rhoptries as a malaria blood stage vaccine antigen. We produced PfRH2-specific mAbs by immunising mice with the 40kDa receptor-binding domain of PfRH2. The PfRH2-specific mAbs cross-reacted with the natural PfRH2 protein present in schizont stage parasites and showed a rhoptry-characteristic staining pattern in immunofluorescence microscopy. However when evaluated in functional in vitro and in vivo assays PfRH2 specific mAbs showed no inhibitory effect on erythrocyte invasion. Furthermore, the invasion-inhibitory effect of mAbs specific for the cysteine-rich protective antigen (PfCyRPA) was not enhanced by PfRH2-specific mAbs.