Lei S, Raftery M A, Conti-Tronconi B M
Department of Biochemistry, University of Minnesota, St. Paul 55108.
Biochemistry. 1993 Jan 12;32(1):91-100. doi: 10.1021/bi00052a013.
Monoclonal antibodies (mAbs) were derived from mice immunized with synthetic peptide sequence regions of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo electric tissue (TAChR). Sequence-specific mAbs were obtained against the following peptides: alpha 1-20, alpha 291-308, alpha 304-322, alpha 332-350, alpha 346-364, alpha 360-378, alpha 376-393, alpha 390-409, and alpha 420-437. The ability of mAbs to recognize native TAChR was quantitated by immunoprecipitation of TAChR solubilized in the nondenaturing detergent Triton X-100. mAbs against peptide alpha 304-322, alpha 332-350, and alpha 360-378 cross-reacted with most or all Triton-solubilized TAChR molecules and, in immunoelectron microscopy experiments, bound to the cytoplasmic surface of AChR-rich postsynaptic membrane fragments. Two mAbs specific for the sequence alpha 376-393, proposed to form an amphypathic alpha helix possibly involved in formation of the ion channel, recognized only approximately 35% of Triton-solubilized TAChR molecules and did not react with membrane-bound TAChR. All of these sequence-specific antibodies recognized SDS-denatured TAChR alpha subunit in Western blots. MAbs specific for the amino-terminal sequence region of the alpha subunit, alpha 1-20, and for the sequences alpha 291-308, alpha 346-364, and alpha 390-409 did not recognize native TAChR. A mAb directed against the carboxyl-terminal region, alpha 420-437, recognized with low apparent titer Triton-solubilized TAChR, not membrane-bound TAChR. In conclusion, a complex membrane protein, TAChR, contains several continuous sequence segments exposed on the TAChR surface, because different mAbs raised against certain synthetic sequences recognized most or all native TAChR molecules. By analogy, it should be possible for most proteins of known sequence to raise anti-peptide antibodies fully cross-reactive with the native cognate protein.
单克隆抗体（mAb）来源于用来自电鳐电组织的烟碱型乙酰胆碱受体α亚基的合成肽序列区域免疫的小鼠。获得了针对以下肽的序列特异性单克隆抗体：α1 - 20、α291 - 308、α304 - 322、α332 - 350、α346 - 364、α360 - 378、α376 - 393、α390 - 409和α420 - 437。通过对溶解在非变性去污剂Triton X - 100中的TAChR进行免疫沉淀来定量单克隆抗体识别天然TAChR的能力。针对肽α304 - 322、α332 - 350和α360 - 378的单克隆抗体与大多数或所有Triton溶解的TAChR分子发生交叉反应，并且在免疫电子显微镜实验中，与富含AChR的突触后膜片段的胞质表面结合。两种对序列α376 - 393特异的单克隆抗体，该序列被认为形成了可能参与离子通道形成的两亲性α螺旋，仅识别大约35%的Triton溶解的TAChR分子，并且不与膜结合的TAChR反应。所有这些序列特异性抗体在蛋白质印迹中都能识别SDS变性的TAChRα亚基。对α亚基的氨基末端序列区域α1 - 20以及序列α291 - 308、α346 - 364和α390 - 409特异的单克隆抗体不能识别天然TAChR。一种针对羧基末端区域α420 - 437的单克隆抗体以低表观滴度识别Triton溶解的TAChR，而不识别膜结合的TAChR。总之，一种复杂的膜蛋白TAChR包含几个暴露在TAChR表面的连续序列片段，因为针对某些合成序列产生的不同单克隆抗体识别大多数或所有天然TAChR分子。以此类推，对于大多数已知序列的蛋白质来说，应该有可能产生与天然同源蛋白完全交叉反应的抗肽抗体。
