Development of monoclonal antibodies to erythroid progenitors in feline bone marrow

Feline bone marrow cells can be enriched for erythroid and myeloid progenitors by counterflow centrifugal elutriation (CCE) and subsequent treatment of the CCE fractions with the soybean agglutinin (SBA) lectin. Separation of CCE fractions into SBA(-) and SBA(+) populations yielded cells enriched for BFU-E and CFU-GM progenitors, respectively. Differential analyses revealed a high percentage of erythroid lineage cells in the SBA(-) fractions and in the SBA(+) fractions a high concentration of myeloid cells of varying maturation stages. The latter cells, but not the CFU-GM progenitors, could be removed by immunomagnetic depletion from CCE fractions using a monoclonal antibody (MAb) specific for CD13 cells in feline bone marrow, resulting also in a population containing predominantly erythroid differentiating cells. Mice were immunized with CCE fractions enriched for erythroid lineage cells and the splenocytes fused with SP2/O cells for hybridoma development. Supernatant culture fluids from 400 hybridomas were analyzed by flow cytometry with whole bone marrow and select CCE/SBA fractions as the target cells. Those hybridomas suggestive of containing the desired antibodies as indicated by the percentage staining were subcloned and the MAbs utilized in clonogenic assays. Treatment of bone marrow cells or CCE fractions with the MAbs followed by immunomagnetic depletions led to identification of two, K-1 and Q-3, reactive with the BFU-E progenitor and one, K-7, reactive only with late-differentiating erythroid lineage cells. Thus, removal of cells from a CCE/SBA suspension reactive with K-1 or Q-3 led to greater than 80% reductions of the BFU-E progenitors and a population enriched for CFU-GM. Removal of cells reactive with MAb K-7, however, led to a marked enrichment, 5-8-fold, of both BFU-E and CFU-GM progenitors.