Anti-Human CD18/CR3 - Dylight® 488 (C565-GRP)

Integrins are a large family of α/β heterodimeric transmembrane molecules that mediate adhesion, migration, cell survival, and cell differentiation1. The leukocytic cell-adhesion molecule (Leu-CAM) family has three members, LFA-1 (CD18/CD11a), CR3 (CD18/CD11b), and p150,95 (CD18/CD11c), all of which share the β chain CD182. KIM127 recognizes a temperature sensitive epitope on CD18, binding to lymphocytes that express LFA-1 as well as to immunopurified CR33. KIM127 promotes LFA-1 and CR3-dependent adhesion events and enhances aggregation of the human B lymphoblastoid line JY and human T cell line MOLT-4. This aggregation is completely blocked by anti-LFA-1 antibody DA36. KIM127 is not associated with increased surface expression of CR3 or LFA-1. KIM127 recognizes residues Gly 504, Leu 506, and Tyr 508 of CD18 in a cysteine-rich repeat located in the long loop protruding from the C-terminal end of the integrin-epidermal growth factor-like (I-EGF) 2 domain4,5,6. The epitope is near the extreme bend (genu) between the integrin headpiece and stalk, buried deep in the crevice formed by the bend between the α subunit thigh and calf-1 domains, and is only accessible when the integrin is extended in its activated conformation6. In resting integrins, the KIM127 epitope is shielded by the ⍺ subunit5. KIM127 preferentially reacts with activated β2 integrins and free β2 subunit compared with resting β2 integrins. A switchblade-like action that extends the headpiece of the integrin away from the plasma membrane is thought to expose the KIM127 epitope when activated6. Activation of LFA-1 ligand binding by Mn2+, and to a lesser degree by Mg2+ in the absence of Ca2+, is positively correlated with increased expression of the KIM127 epitope5. PMA also induces a small but consistent increase in KIM127 expression. KIM127 was obtained from a BALB/c mouse immunized with CR3 immunopurified from a human white cell lysate3. Hybridoma lines were created by fusing spleen cells with Sp2/0 cells and screening by ELISA for their ability to bind to immunopurified CR3 and then for their ability to promote aggregation of different cell types. Mildly denaturing conditions are required during Western blotting because boiling the sample destroys the KIM127 epitope.