A sensitive assay for detecting low‐affinity interactions at the cell surface reveals no additional ligands for the adhesion pair rat CD2 and CD48

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Abstract
The ligand for the T cell antigen CD2 is CD48 in rodents, but CD58 in humans. The extracelluar parts of these three antigens are structurally related in that all contain two immunoglobulin superfamily (IgSF) domains. There have been reports of alternative ligands for CD2 in the human, but not so far in rodents. We describe the analysis of ligands for rat CD2 and CD48 using fluorescent beads capable of displaying a high ligand density and detecting low‐affinity interactions like that of CD2 with CD48 (Kd = 60 − 90 μM). Monovalent chimeric proteins containing the two IgSF domains of rat CD48 or CD2 and domains 3 and 4 of rat CD4 (CD4d3+4) were anchored to fluorescent covaspheresTMvia a CD4 monoclonal antibody (mAb) with the CD48 or CD2 domains available for ligand binding. Multivalent CD48‐CD4d3 + 4 covaspheresTMgave strong specific binding to rat CD2 expressed on the surface of transfected Jurkat cells. CD48‐CD4d3+4 was compared with CD48‐IgG and CD48‐IgM as tools for detecting binding at the cell surface. Soluble divalent CD48‐IgG and decavalent CD48‐IgM bound to soluble CD2 with a Koffof around 10−3s−1as determined using a BIAcoreTMbiosensor. However, binding to cells by CD48‐IgG and CD48‐IgM was only detectable when they were immobilized on covaspheresTMand represented no increase in sensitivity over CD48‐CD4 covaspheresTMwhen tested for binding to cells expressing high and low levels of CD2. CD48‐CD4d3 + 4 covaspheresTMonly bound to rat cells expressing CD2. In the reverse orientation, binding of CD2‐CD4d3 + 4 covaspheresTMwas dependent on expression of CD48. Pre‐incubation of cells with CD2 or CD48 mAb abolished all binding of CD48‐CD4d3 + 4 or CD2‐CD4d3 + 4, respectively. The data provide no evidence for an alternative ligand for rat CD2 or CD48.