Development of a cell-free binding assay for rat ICAM-1/LFA-1 interactions using a novel anti-rat LFA-1 monoclonal antibody and comparison with a cell-based assay

Abstract

The importance of the interaction between lymphocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) in the progression of inflammatory responses in vivo has been demonstrated mainly in rats. The present study was undertaken to develop binding assays suitable for measuring the rat ICAM-1/LFA-1 interaction in vitro. We first examined binding of rat T lymphoma FTL43 cells, which express LFA-1, to immobilized rat ICAM-1. Although FTL43 cells bound avidly to immobilized ICAM-1 and the binding was abolished with anti-LFA-1 monoclonal antibodies (mAbs), the binding was not completely inhibited by most anti-ICAM-1 mAbs. We next purified rat LFA-1 from FTL43 cells and constructed a cell-free binding assay. By using a newly developed anti-rat LFA-1 mAb RL14/9, which does not inhibit ICAM-1/LFA-1 interactions, binding of purified rat LFA-1 to immobilized ICAM-1 was successfully detected, whereas only a low signal to noise ratio was observed when binding of ICAM-1 to immobilized LFA-1 was examined. Moreover, we found that simultaneous addition of purified LFA-1 and biotinylated RL14/9 to ICAM-1-coated wells resulted in more sensitive detection of rat ICAM-1/LFA-1 binding. The binding was completely blocked with both anti-LFA-1 and anti-ICAM-1 mAbs and was much more sensitive to inhibition by the ICAM-1-IgG chimera, as compared with the cell-based assay. These results indicate that the cell-free binding assay provides a rapid and sensitive method for screening rat ICAM-1/LFA-1 antagonists, whose therapeutic effect on inflammatory diseases can further be evaluated in vivo.