PRODUCTION AND IMMUNOHISTOCHEMICAL APPLICATION OF MONOCLONAL-ANTIBODIES AGAINST DELTA SLEEP-INDUCING PEPTIDE
PRODUCTION AND IMMUNOHISTOCHEMICAL APPLICATION OF MONOCLONAL-ANTIBODIES AGAINST DELTA SLEEP-INDUCING PEPTIDE
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DOI：
10.1016/0891-0618(92)90005-b
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发表时间：
1992-11-01
影响因子：
2.8
通讯作者：
BOURAS, C
中科院分区：
文献类型：
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作者：
CHARNAY, Y;GOLAZ, J;BOURAS, C
用delta - sleep-including peptide （DSIP）免疫大鼠制备单克隆抗体。将大鼠脾细胞与骨髓瘤SP2/0细胞系融合。采用点免疫法结合DSIP和部分DSIP片段，筛选杂交瘤上清液。6个稳定产菌克隆的上清液与DSIP有反应。结果表明，这些单克隆抗体均能识别DSIP五羧基末端的表位（DSIP5-9）。将这些单克隆抗体应用于大鼠正中隆起切片，对主要定位于外侧区域的大量纤维和终末样结构进行了强有力的免疫标记。DSIP单克隆抗体和LHRH多克隆抗血清洗脱实验显示，免疫反应模式几乎完全重叠。尽管在小鼠、仓鼠和沙鼠的正中隆起中也很容易发现许多lhrh免疫反应性神经元元件，但用DSIP单克隆抗体孵育的切片未能产生任何免疫反应。综上所述，这些数据证明了DSIP/LHRH免疫标记系统的独立性。此外，研究表明，在大鼠中隆起中检测到的dsip5 -9相关表位在其他三种啮齿类动物中没有对应的表位。这些物种差异可能反映了最初在家兔中发现的非肽DSIP的羧基末端序列在所有啮齿动物中并不保守。
Monoclonal antibodies were produced following immunization of rats with delta sleep-including peptide (DSIP). The spleen cells of the rats were fused with the myeloma cell line SP2/0. The supernatants of hybridomas were screened on a solid-phase immunoassay using dot-immunobinding of DSIP and some DSIP fragments. The supernatants of six stable producer clones were found to react with DSIP. From this procedure it was also deduced that all these monoclonal antibodies recognized epitope(s) of the penta carboxy-terminal region of DSIP (DSIP5-9). Application of these monoclonal antibodies to rat median eminence sections gave a strong immunolabelling of a large population of fibres and terminal-like structures, mainly localized through the lateral areas. Elution-restaining experiments using a monoclonal antibody to DSIP and a polyclonal antiserum to luteinizing hormone-releasing hormone (LHRH) showed that the patterns of immunoreactivity respectively visualized overlap almost completely. Although numerous LHRH-immunoreactive neuronal elements were also easily demonstrated in the median eminence of the mouse, the hamster and the gerbil species, incubation of sections with monoclonal antibodies to DSIP failed to give any immunoreaction. Taken together these data argue for the independence of the DSIP/LHRH immunolabelling systems. Furthermore, it was demonstrated that DSIP5-9-related epitopes detected in the rat median eminence have no counterpart in the three other rodent species investigated. These species differences may reflect the fact that the carboxy-terminal sequence of the nonapeptide DSIP originally discovered in the rabbit is not conserved in all rodent species.