MONOCLONAL ANTIBODY ANALYSIS OF MUSCLE FIBER TYPES

已经用常规抗血清进行了免疫组织化学研究， 检查肌肉纤维类型及其变化，但结果相互矛盾 获得了 我们计划研究纤维类型的属性， 通过该技术产生的肌球蛋白同工酶的单克隆抗体（MAb's） 科勒和米尔斯坦（1975）的结果。 为此目的， 将使用适当的肌球蛋白产生慢肌球蛋白的轻链 从鸟类和哺乳动物来源作为免疫原。 (Some有趣 已经产生了针对前肌球蛋白的单克隆抗体， 鸡的背阔肌）。 特异性和约束力 单克隆抗体与各种肌球蛋白同形物的亲和力将是 用放射免疫法和免疫荧光法进行比较。 位置 重链（及其亚片段）和轻链中的抗原决定簇 链将通过抗体结合试验和超微结构测定 免疫细胞化学 抗快速肌球蛋白轻链和重链的单克隆抗体 已经被生产出来并在费施曼博士的 实验室;他们将提供本研究。 使用2套 鸡的良好表征的抗体纤维类型分析， 哺乳动物肌肉将通过免疫荧光在冷冻切片上进行。 希望纤维类型和亚类型的分类将有助于 肌球蛋白同工酶分布的基础上建立的。 手术 鸡和大鼠肌肉的去神经支配将在新生儿和 成人阶段 不同的敏感性去神经支配的快， 慢纤维的分化和维持， 评价肌球蛋白同工酶。 这些研究直接关系到 肌肉神经源性疾病 由于交叉反应性的 抗体与人类肌肉（我们的几个单克隆抗体的给予强 用活组织检查的人样品的免疫荧光），产生的单克隆抗体 在这个项目中也应该是有价值的正常和 患病的人体肌肉

Immunohistochemical studies with conventional antisera have been done to examine muscle fiber types and their alterations but conflicting results were obtained. We plan to investigate the properties of fiber types using monoclonal antibodies (MAb's) to myosin isozymes produced by the technique of Kohler and Milstein (1975). For this purpose antibodies to heavy and light chains of slow myosins will be produced using appropriate myosins from both avian and mammalian sources as immunogens. (Some interesting monoclonals have already been generated against the myosin of anterior latissimus dorsi muscle of the chicken). The specificities and binding affinities of the MAb's with respect to various myosin isomorphs will be compared by radioimmune assay and immunofluoresence. Location of the antigenic determinants in heavy chain (and its subfragments) and light chains will be determined by antibody binding assays and ultrastructural immunocytochemistry. MAb's against light and heavy chains of fast myosins have already been produced and are being characterized in Dr. Fischman's laboratory; they will be available for the present study. Using the 2 sets of well characterized antibodies fiber type analysis of chicken and mammalian muscles will be done on frozen sections by immunofluorescence. It is hoped that classification of fiber types and sub-types will be established on the basis of myosin isozyme distribution. Surgical denervation of chicken and rat muscles will be done at both neonatal and adult stages. The differential susceptibility to denervation of fast and slow fibers in respect to both differentiation and maintenance of their myosin isozymes will be evaluated. These studies are directly relevant to neurogenic diseases of muscle. Because of the cross-reactivity of the antibodies with human muscle (several of our MAb's give strong immunofluorescence with biopsied human samples), the monoclonals generated in this project should also be valuable in fiber typing of normal and diseased human muscles.