A Monoclonal Antibody Recognizing K- but Not γ- and δ-Opioid Receptors |
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Authors: | Katalin Maderspach Klára Németh József Simon Sándor Benyhe Mária Szücs Mária Wollemann |
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Institution: | Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged. |
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Abstract: | 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) |
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Keywords: | k-Opioid receptor Monoclonal antibody Brain (frog chick rat guinea pig) |
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