DETERMINATION OF THE BINDING-AFFINITY OF AN ANTI-CD34 SINGLE-CHAIN ANTIBODY USING A NOVEL, FLOW-CYTOMETRY BASED ASSAY

A single chain antibody (scFv) was constructed from a hybridoma expres sing the anti-CD34 monoclonal antibody My10. The scFv was expressed in the mouse fibroblast cell line NIH 3T3, and purified from culture sup ernatant via an epitope tag fused to the C-terminus of the protein. Th e scFv equilibrium dissociation constant (K-D) was determined to be 2. 4 x 10(-7) M using a fluorescence based flow cytometry assay involving recognition of the epitope tag, and bound approximately 24-fold less avidly to CD34 expressing KG-1a cells than the native antibody My10. T his novel and previously unreported method for determining antibody bi nding affinity offers several advantages over alternative methods. It is rapid and simple, and unlike methods that directly label the antibo dy, it involves no covalent modifications of antibody variable domain residues that could potentially interfere with antigen binding. The K- D for the anti-CD33 antibody HuG1 (Caron et al. (1992) The biological and immunological features of humanized M195 (anti-CD33) monoclonal an tibodies. Cancer Res. 52, 6761-6767) was determined as well. The close agreement of this value and the previously reported value, determined by a radioligand competition method, validates the use of this assay for antibody affinity determination. We discuss various potential appl ications for this anti-CD34 scFv.