O'Brien M A, Katahira E J, Flanagan T R, Arnold L W, Haughton G, Bollenbacher W E
Department of Biology, University of North Carolina, Chapel Hill 27514.
J Neurosci. 1988 Sep;8(9):3247-57. doi: 10.1523/JNEUROSCI.08-09-03247.1988.
The prothoracicotropic hormone (PTTH) is an insect cerebral peptide that stimulates the prothoracic glands to produce the steroid hormone ecdysone thus initiating molting and metamorphosis. "Big" PTTH, one of several molecular forms of the neurohormone, was isolated from brains of the tobacco hornworm Manduca sexta, and fractionated by high-pressure liquid chromatography (HPLC) for use in antibody production. A murine polyclonal antiserum and a monoclonal antibody (MAb) have been generated using this highly purified preparation of big PTTH. Antisera and hybridoma supernatants were screened with an indirect, brain whole-mount immunocytological assay, and antibody specificity was confirmed by immunocytological, ELISA, and functional criteria. In brain whole-mount preparations, the MAb (A2H5) and antiserum specifically immunostained the lateral protocerebral neurosecretory cells (L-NSC III), the prothoracicotropes, which produce PTTH. This immunostaining was blocked by preadsorbing the antibodies with big PTTH. Analysis of the elution of HPLC-fractionated big PTTH with an in vitro bioassay for the neurohormone and an ELISA employing the A2H5 MAb resulted in peaks of activity that were superimposable. Finally, the antiserum and A2H5 MAb inhibited big PTTH activation of the prothoracic glands to synthesize ecdysone in the in vitro bioassay for the neurohormone. With these specific antibodies, the organization of the PTTH neuroendocrine axis has been defined. It is now evident that both of the peptidergic neurons that comprise the L-NSC III are prothoracicotropes, and that the corpora allata are the neurohemal organs for the release of big PTTH into the hemolymph. This study indicates that these specific antibodies will be useful in investigations of numerous aspects of the biology of this cerebral neuroendocrine axis.
促前胸腺激素（PTTH）是一种昆虫脑肽，它刺激前胸腺产生类固醇激素蜕皮激素，从而启动蜕皮和变态过程。“大”PTTH是这种神经激素的几种分子形式之一，从烟草天蛾烟草天蛾的脑中分离出来，并通过高压液相色谱（HPLC）进行分级分离，用于制备抗体。使用这种高度纯化的大PTTH制剂产生了鼠多克隆抗血清和单克隆抗体（MAb）。通过间接脑全装免疫细胞测定法筛选抗血清和杂交瘤上清液，并通过免疫细胞、ELISA和功能标准确认抗体特异性。在脑全装制剂中，MAb（A2H5）和抗血清特异性免疫染色外侧原脑神经分泌细胞（L-NSC III），即产生PTTH的促前胸腺激素分泌细胞。用大PTTH预吸附抗体可阻断这种免疫染色。用神经激素的体外生物测定法和使用A2H5 MAb的ELISA分析HPLC分级分离的大PTTH的洗脱，得到了可叠加的活性峰。最后，在神经激素的体外生物测定中，抗血清和A2H5 MAb抑制了大PTTH对前胸腺合成蜕皮激素的激活作用。有了这些特异性抗体，PTTH神经内分泌轴的组织已经明确。现在很明显，组成L-NSC III的两个肽能神经元都是促前胸腺激素分泌细胞，并且咽侧体是将大PTTH释放到血淋巴中的神经血器官。这项研究表明，这些特异性抗体将有助于研究这个脑神经内分泌轴生物学的许多方面。
