Yang Tangbin, Wang Jianhe, Qu Lina, Zhong Ping, Yuan Yanhong
Laboratory of Space Cellular and Molecular Biology, Institute of Space Medico-Engineering, Beijing.
J Pineal Res. 2006 May;40(4):350-4. doi: 10.1111/j.1600-079X.2006.00314.x.
Anti-melatonin monoclonal antibodies (MAbs) of high titer were prepared by coupling melatonin to bovine serum albumin with formaldehyde and by immunizing BALB/c mice with multifocal intradermal injections and by fusing high titer antibody producing spleen cells with myeloma cells of SP2/0 origin. Five MAbs were selected for further characterization as classes and subclasses. After four successive limiting dilutions, antibodies were produced by these five clones with high affinities ranging from 10(9) to 10(11)/m. These clones were found to be of the immunoglobulin Ig G1 and IgG(2b) subclass with kappa light chain. A systematic study of cross-reactions with seven compounds (indole, aromatic and imidazole derivatives) showed that the antibody had a high specificity for melatonin, low reactivity with 6-hydroxymelatonin and N-acetyl-5-hydroxytryptamine, and no detectable reactivity with tryptamine, l-tryptophan, 5-methoxytryptamine and N-acetyl-L-tryptophan. The roles of the indole nucleus and the side chain in the determination of the antigenic properties of the molecule are discussed. One of the MAbs, 4C9D7, was used to establish a competitive enzyme-linked immunosorbent assay for the detection of melatonin in supernatant.
通过用甲醛将褪黑素与牛血清白蛋白偶联、对BALB/c小鼠进行多部位皮内注射免疫以及将产生高滴度抗体的脾细胞与SP2/0来源的骨髓瘤细胞融合，制备了高滴度的抗褪黑素单克隆抗体（MAbs）。选择了五种MAbs进行进一步的类别和亚类鉴定。经过连续四次有限稀释后，这五个克隆产生了亲和力范围为10⁹至10¹¹/m的高亲和力抗体。发现这些克隆属于具有κ轻链的免疫球蛋白Ig G1和IgG(2b)亚类。对与七种化合物（吲哚、芳香族和咪唑衍生物）的交叉反应进行的系统研究表明，该抗体对褪黑素具有高特异性，与6-羟基褪黑素和N-乙酰-5-羟色胺的反应性较低，与色胺、L-色氨酸、5-甲氧基色胺和N-乙酰-L-色氨酸无可检测的反应性。讨论了吲哚核和侧链在确定分子抗原特性中的作用。其中一种MAb，4C9D7，用于建立一种竞争性酶联免疫吸附测定法，用于检测上清液中的褪黑素。
