Malouf N N, Coronado R, McMahon D, Meissner G, Gillespie G Y
Proc Natl Acad Sci U S A. 1987 Jul;84(14):5019-23. doi: 10.1073/pnas.84.14.5019.
In skeletal muscle, dihydropyridine receptors and dihydropyridine-sensitive Ca2+ channels are preferentially localized in the transverse tubular membranes. Starting with an antigenic membrane fraction enriched in rabbit muscle transverse tubules (T-tubules), several monoclonal antibodies were produced by a fusion of spleen cells from an immunized BALB/c mouse with P3 X 63Ag.8.6.5.3 mouse myeloma cells. Antibodies were screened according to a scheme designed to select IgG immunoglobins that recognized a determinant specifically associated with the T-tubule membrane. Antibodies that fulfilled the screening criteria were used in in vitro planar bilayer recording of the activity of the dihydropyridine-sensitive Ca2+ channel present in T-tubules. Cells producing one antibody (Ab 21) survived cloning dilution and stably produced a monoclonal antibody (mAb21-4) that increased the rate of single channel opening when interacting with the internal side of the channel protein. mAb21-4 immobilized by covalent crosslinking on beads (Affi-Gel 10) consistently immunoprecipitated polypeptide bands with the following electrophoretic mobility: Mr values of greater than or equal to 175,000; 90,000; 55,000; and 34,000.
在骨骼肌中，二氢吡啶受体和对二氢吡啶敏感的Ca2+通道优先定位于横管膜。从富含兔肌肉横管（T管）的抗原性膜组分开始，通过将免疫的BALB/c小鼠的脾细胞与P3 X 63Ag.8.6.5.3小鼠骨髓瘤细胞融合，产生了几种单克隆抗体。根据设计的方案筛选抗体，以选择识别与T管膜特异性相关的决定簇的IgG免疫球蛋白。满足筛选标准的抗体用于体外平面双层记录T管中存在的对二氢吡啶敏感的Ca2+通道的活性。产生一种抗体（Ab 21）的细胞在克隆稀释后存活，并稳定产生一种单克隆抗体（mAb21-4），该抗体与通道蛋白的内侧相互作用时会增加单通道开放率。通过共价交联固定在珠子（Affi-Gel 10）上的mAb21-4始终免疫沉淀出具有以下电泳迁移率的多肽条带：Mr值大于或等于175,000；90,000；55,000；和34,000。
