A Monoclonal Antibody Recognizing K ‐ but Not γ‐ and δ‐Opioid Receptors

Abstract: A monoclonal antibody (mAb), KA8, that interacts with the k ‐opioid receptor binding site was generated. BALB/ c female mice were immunized with a partially purified k ‐opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibodyproducing hybridomas were screened for competition with opioid ligands in a modified enzyme‐linked immunosorbent assay. The cell line KA8 secretes an IgG1 ( k ‐light chain) immunoglobulin. The mAb KA8 purified by affinity chromatography on protein A‐Sepharose CL4B was able to precipitate the antigen from a solubilized and affinity‐purified frog brain k ‐opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [ 3 H]ethylketocyclazocine ([ 3 H]EKC) binding to the frog brain membrane fraction in a concentration‐dependent manner to a maximum to 72%. The degree of the inhibition was increased to 86% when γ‐and δ‐opioid binding was suppressed by 100 n M [D‐Ala 2 ,NMe‐Phe 4 ,Gly‐ol]‐enkephalin (DAGO) and 100 n M [D‐Ala 2 ,L‐Leu 5 ]‐enkephalin (DADLE), respectively, and to 100% when γ‐, δ‐, and k 2 ‐sites were blocked by 5 γ M DADLE. However, the γ‐specific [ 3 H]DAGO and the δ‐preferring [ 3 H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the k ‐ but not the γ‐ and δ‐subtype of opioid receptors. The mAb KA8 also inhibits specific [ 3 H]naloxone and [ 3 H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [ 3 H]EKC binding to guinea pig cerebellar membranes. These findings suggest homologies in the k ‐opioid binding site of frog brain and rat brain as well as chick neurons, but the k ‐opioid receptor subtype in the guinea pig cerebellum may be different.