Johnston M I, Imai J, Lesiak K, Jacobsen H, Sawai H, Torrence P F
Biochemistry. 1985 Aug 13;24(17):4710-8. doi: 10.1021/bi00338a033.
To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.
为了确定与抗寡核苷酸抗体结合相关的表位，建立了几种产生针对2',5'-寡腺苷酸的单克隆抗体的杂交瘤。一种采用包被有Ficoll-2',5'-寡腺苷酸缀合物的微量滴定孔的固相酶联免疫测定法，被证明可用于筛选和鉴定杂交瘤上清液。对照实验表明，在该测定所采用的条件下，缀合物不可逆地吸附于聚苯乙烯孔上。通过竞争测定法测量单克隆抗体与众多2',5'-寡腺苷酸类似物的反应性。源自用相同或不同免疫原免疫的不同小鼠的几种单克隆抗体具有独特的精细特异性。至少一个2',5'-磷酸二酯键在形成每个表位中很重要，这表明核糖磷酸主链是定义寡核苷酸抗原结构域的关键要素。嘌呤碱基也很重要，碱基的修饰对抗体识别程度有不同影响。寡核苷酸的长度和末端性质也具有一定重要性。在一些情况下，2',5'-寡腺苷酸与载体蛋白连接产生的修饰也有助于形成决定簇。对2',5'-寡腺苷酸最具特异性的单克隆抗体对离子强度相对不敏感。相比之下，具有2',5'-寡嘌呤特异性的单克隆抗体似乎通过一个离子对结合2',5'-寡腺苷酸，而具有低碱基特异性的单克隆抗体的结合似乎通过两个离子对。结果表明，2',5'-连接的寡腺苷酸-蛋白质复合物具有至少三个不同的与寡核苷酸相关的抗原表面，可被单克隆抗体以高表观亲和力识别。本文提出了这三个表位的模型。
