Lazarus L H, Irons B J, Grimes L M, Wilson W E, Guglietta A, Yajima H
Peptide Neurochemistry Group, LBNT, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
J Neurosci Methods. 1988 Mar;23(2):161-72. doi: 10.1016/0165-0270(88)90188-4.
Five unique, high affinity rabbit polyclonal antibodies against neuromedin B were characterized in a radioimmunoassay in terms of the following parameters: pH and type of buffer, ionic strength, and non-ionic detergents in order to optimize immunoglobulin-peptide interaction; specificity using peptides of the bombesin family, in addition to the tachykinin substance P; and affinity to neuromedin B. Optimum conditions included acidic pH (5.25), high ionic strength (greater than 0.1 M) and absence of non-ionic detergents, which inhibited the assay. Affinities for the 5 antibodies ranged from 10 to 48 fmol neuromedin B with titers from 1:1,000 to 1:10,000 and the sequence-specificity covered the entire peptide; cross-reactivity towards substance P was negligible. As a model tissue, rat spinal cord was homogenized with 5 different extraction solvents, including acetone, methanol, acid and alkaline conditions, and assayed by each polyclonal antiserum; neuromedin B immunoreactivity levels were highest in acid and alkaline extracts and reflected the specificity of the antibody used. Applying these antisera to rat brain extracts, the posterior pituitary gland contained the highest concentration of immunoreactive equivalents of neuromedin B followed by the anterior pituitary, hypothalamus, and hippocampus. The immunoreactive content in the pituitary and hypothalamus, however, depended on the particular antisera used with significant (P less than 0.01) differences existing between them. Further application of these polyclonal antibodies to a spinal cord extract analyzed by isocratic reverse-phase HPLC conditions also revealed differences in their cross-reactivity with the immunoreactive peptides. These antisera may now be used as molecular probes for the determination of extractable immunoreactive neuromedin B from neural tissue and in situ localization by immunohistochemical techniques.
针对神经降压素B制备了5种独特的、高亲和力的兔多克隆抗体，并在放射免疫分析中对以下参数进行了表征：用于优化免疫球蛋白 - 肽相互作用的缓冲液的pH值和类型、离子强度以及非离子去污剂；除速激肽P物质外，使用蛙皮素家族肽段检测其特异性；以及对神经降压素B的亲和力。最佳条件包括酸性pH值（5.25）、高离子强度（大于0.1 M）且不存在抑制该分析的非离子去污剂。这5种抗体的亲和力范围为10至48 fmol神经降压素B，效价为1:1000至1:10000，序列特异性覆盖整个肽段；对P物质的交叉反应可忽略不计。以大鼠脊髓作为模型组织，用5种不同的提取溶剂（包括丙酮、甲醇、酸性和碱性条件）进行匀浆，并使用每种多克隆抗血清进行检测；神经降压素B免疫反应性水平在酸性和碱性提取物中最高，反映了所用抗体的特异性。将这些抗血清应用于大鼠脑提取物，发现垂体后叶中神经降压素B免疫反应性当量的浓度最高，其次是垂体前叶、下丘脑和海马体。然而，垂体和下丘脑中的免疫反应性含量取决于所用的特定抗血清，它们之间存在显著（P小于0.01）差异。将这些多克隆抗体进一步应用于通过等度反相高效液相色谱条件分析的脊髓提取物，也揭示了它们与免疫反应性肽段的交叉反应存在差异。这些抗血清现在可用作分子探针，通过免疫组织化学技术测定神经组织中可提取的免疫反应性神经降压素B并进行原位定位。
