Generation of Novel Monoclonal Antibodies Recognizing Rabbit CD34 Antigen

The rabbit is a widely used experimental model for human translational research and stem cell therapy. Many studies have focused on rabbit mesenchymal stem cells from different biological sources for their possible application in regenerative medicine. However, a minimal number of studies have been published aimed at rabbit hematopoietic stem/progenitor cells, mainly due to the lack of specific anti-rabbit CD34 antibodies. In general, CD34 antigen is commonly used to identify and isolate hematopoietic stem/progenitor cells in humans and other animal species. The aim of this study was to develop novel monoclonal antibodies highly specific to rabbit CD34 antigen. We used hybridoma technology, two synthetic peptides derived from predicted rabbit CD34 protein, and a recombinant rabbit CD34 protein as immunogens to produce monoclonal antibodies (mAbs) specific to rabbit CD34. The produced antibodies were screened for their binding activity and specificity using ELISA, flow cytometry, and Western blot analysis. Finally, four mAbs (58/47/26, 58/47/34, 182/7/80, and 575/36/8) were selected for the final purification process. The purified mAbs recognized up to 2-3% of total rabbit bone marrow cells, while about 2% of those cells exhibited CD45 expression, which are likely rabbit primitive hematopoietic stem cells and their hematopoietic progenitors, respectively. The newly generated and purified mAbs specifically recognize CD34 antigen in rabbit bone marrow or peripheral blood and can be therefore used for further immunological applications, to study rabbit hematopoiesis or to establish a new animal model for hematopoietic stem cell transplantation studies.