Abstract
The aim of this study was to establish a method to obtain antibodies against cell-surface molecules of hematopoietic stem and progenitor cells in situ. A phage-displayed scFv antibody library with a size of 2 × 106 clones was constructed from spleens of mice immunized with living KG1a human leukemia cells. Living cell panning was used to screen anti-KG1a cell antibody fragments. After four rounds of panning, 27 out of 126 scFv fragments showed detectable binding to KG1a cells without cross-reaction to non-hematopoietic cell lines. Individual clones were analyzed by cell-based ELISA and flow cytometry for their binding specificity. Their sequence analysis showed highest homology to mouse gamma heavy chain subgroup II and mouse Kappa subgroup III genes, with four amino acids difference in VH and identical VL. Further, a fusion protein of scFv 5C1 to core-streptavidin was cloned and produced. It was used to search for cell-surface antigen on immunoblots and to test its effect on KG1a cell growth in cell culture. The scFv5C1::core-streptavidin fusion protein recognized a molecule with MW 85/125 KDa in immunoblots of KG1a cell membrane proteins and inhibited KG1a cell homoaggregation in cell cultures. The results validate an efficient approach of making antibodies against living cells and searching for functional inhibitors of cell-surface molecules.
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