A monoclonal antibody recognizing kappa- but not mu- and delta-opioid receptors.
A monoclonal antibody (mAb), KA8 that interacts with the kappa-opioid receptor binding site was generated. BALB/c female mice were immunized with a partially purified kappa-opioid receptor preparation from frog brain. Spleen cells were hybridized with SP2/0AG8 myeloma cells. The antibody-producing hybridomas were screened for competition with opioid ligands in a modified enzyme-linked immunosorbent assay. The cell line KA8 secretes an IgG1 (kappa-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 kappa-opioid receptor preparation. In competition studies, the mAb KA8 decreased specific [3H]ethylketocyclazocine ([3H]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 mu- and delta-opioid binding was suppressed by 100 nM [D-Ala2,NMe-Phe4,Gly-ol]-enkephalin (DAGO) and 100 nM [D-Ala2,L-Leu5]-enkephalin (DADLE), respectively, and to 100% when mu-, delta-, and kappa 2-sites were blocked by 5 microM DADLE. However, the mu-specific [3H]DAGO and the delta-preferring [3H]DADLE binding to frog brain membranes cannot be inhibited by mAb KA8. These data suggest that this mAb is recognizing the kappa- but not the mu- and delta-subtype of opioid receptors. The mAb KA8 also inhibits specific [3H]naloxone and [3H]EKC binding to chick brain cultured neurons and rat brain membranes, whereas it has only a slight effect on [3H]EKC binding to guinea pig cerebellar membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Maderspach
K
K
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged.
Németh
K
K
Simon
J
J
Benyhe
S
S
Szücs
M
M
Wollemann
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001202:Ascitic Fluid / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D002642:Chick Embryo; D016314:Ethylketocyclazocine / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D009270:Naloxone / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D011233:Precipitin Tests; D011893:Rana esculenta; D011957:Receptors, Opioid / Q000276:immunology*; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa
D000911:Antibodies, Monoclonal; D011957:Receptors, Opioid; D017465:Receptors, Opioid, delta; D017473:Receptors, Opioid, kappa; D009270:Naloxone; D016314:Ethylketocyclazocine