The affinity of protein-protein interaction in biological systems ranges from extremely strong, such as that between avidin and biotin (Ka = approximately 10 15 M-1) to the very weak, such as E-selectin and E-selectin ligand-1 (Ka = approximately 7.4 × 104 M-1S-1) (1). It is known that the interactions mediated by the surface receptors of immune cells are often of low-affinity and transient in nature (2). Despite weak affinities, cell surface interactions among immune cells have been shown to play an important role in many functions such as surveillance, immunosynapse formation, and activation (2). It is therefore desirable to devise a way to select and enrich cells expressing low-affinity ligands for specific immune cell surface receptors. Here we use the well-studied interaction between CD97 and CD55 as a model system to describe a simple method for this purpose. Both CD97 and CD55 are cell surface proteins expressed predominantly on leukocytes (3–5). The binding of CD97 to CD55 has been demonstrated to be both of low affinity (86 μM) and transient (off-rate; 0.6/s) (6). We and others have previously shown that the interaction is mediated exclusively by the epidermal growth factor (EGF)-like domains of CD97 and the short consensus repeats (SCR) of CD55 in a Ca2+-dependent fashion (6,7). Furthermore, a CD97-like molecule, EMR2, has been shown not to bind CD55 even though it contains highly homologous EGF-like domains (97.5% identity), therefore providing an ideal negative control (8). Our method is based on (i) the efficient isolation and coupling of Fc-fusion proteins to protein AGconjugated paramagnetic beads (Protein A/G Dynabeads®; Dynal A.S., Oslo, Norway) (Figure 1A) and (ii) the simple capture and separation of ligand-positive cells from the Fcfusion protein-coated paramagnetic beads (Figure 1B). The coupling of the Fc-fusion proteins to the protein AG-conjugated paramagnetic beads generates a multivalent protein probe, thereby increasing binding efficiency (Figure 1, A and B). As the major CD55-binding CD97 isoform contains three EGF-domains, we fused the three EGF-domains immediately upstream of the mouse Fc region (Figure 1A). CD97(EGF-1,2,5)-mFc and EMR2(EGF-1,2,5)-mFc fusion proteins were produced as described previously in transiently transfected HEK293T cells (Figure 1C). Protein AG-conjugated paramagnetic beads were washed three times in binding buffer [0.5% bovine serum albumin (BSA) in Hank’s Balanced Salt solution (HBSS)], reconstituted in the binding buffer to the original concentration (10 mg/mL), and incubated at 4°C for 30 min with or without 2 μg mFc-fusion protein/10 μL beads/reaction. Following the removal of unbound proteins by magnetic separation, the fusion protein-bead complexes were then resuspended in 20 μL binding buffer/reaction. K562 cells (CD55+) were washed, adjusted to a cell density of 3 × 106 cells/mL, and incubated at 4°C with the protein-bead