title: 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.
authors.affiliation: New Product Research Laboratories IV, Tokyo R&D Center, Daiichi Pharmaceutical, 1-16-13 Kita-kasai, Edogawa-ku, Tokyo, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Mukasa
authors.forename: A
authors.initials: A
authors.lastname: Satoh
authors.forename: Y
authors.initials: Y
authors.lastname: Tominaga
authors.forename: M
authors.initials: M
authors.lastname: Yamazaki
authors.forename: K
authors.initials: K
authors.lastname: Matsumoto
authors.forename: Y
authors.initials: Y
authors.lastname: Iigo
authors.forename: T
authors.initials: T
authors.lastname: Higashida
authors.forename: Y
authors.initials: Y
authors.lastname: Kita
authors.forename: M
authors.initials: M
authors.lastname: Miyasaka
authors.forename: T
authors.initials: T
authors.lastname: Takashi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002474:Cell-Free System; D005069:Evaluation Studies as Topic; D007118:Immunoassay / Q000379:methods*; D066298:In Vitro Techniques; D007249:Inflammation / Q000209:etiology / Q000276:immunology / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D051381:Rats
chemical_list: D000911:Antibodies, Monoclonal; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1