Maderspach K, Németh K, Simon J, Benyhe S, Szücs M, Wollemann M
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Szeged.
J Neurochem. 1991 Jun;56(6):1897-904. doi: 10.1111/j.1471-4159.1991.tb03446.x.
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)
制备了一种与κ-阿片受体结合位点相互作用的单克隆抗体（mAb）KA8。用从蛙脑部分纯化的κ-阿片受体制剂免疫BALB/c雌性小鼠。脾细胞与SP2/0AG8骨髓瘤细胞杂交。在改良的酶联免疫吸附测定中筛选产生抗体的杂交瘤与阿片样物质配体的竞争情况。细胞系KA8分泌IgG1（κ轻链）免疫球蛋白。通过蛋白A-琼脂糖凝胶CL4B亲和层析纯化的单克隆抗体KA8能够从溶解并亲和纯化的蛙脑κ-阿片受体制剂中沉淀抗原。在竞争研究中，单克隆抗体KA8以浓度依赖方式使与蛙脑膜部分的特异性[³H]乙基酮环唑新（[³H]EKC）结合减少，最大可达72%。当分别用100 nM [D-Ala²，NMe-Phe⁴，Gly-ol]-脑啡肽（DAGO）和100 nM [D-Ala²，L-Leu⁵]-脑啡肽（DADLE）抑制μ-和δ-阿片样物质结合时，抑制程度增加到86%，当用5 μM DADLE阻断μ-、δ-和κ₂位点时，抑制程度增加到100%。然而，单克隆抗体KA8不能抑制[³H]DAGO与蛙脑膜的μ特异性结合以及[³H]DADLE与蛙脑膜的δ偏好性结合。这些数据表明该单克隆抗体识别的是阿片受体的κ亚型而非μ和δ亚型。单克隆抗体KA8还抑制[³H]纳洛酮和[³H]EKC与鸡脑培养神经元和大鼠脑膜的特异性结合，而对[³H]EKC与豚鼠小脑膜的结合只有轻微影响。（摘要截短于250字）
