Lightbody J J, Ziaja E L, Lugo S D, Mainwaring M G, Vinogradov S N, Shishikura F, Walz D A, Suzuki T, Gotoh T
Biochim Biophys Acta. 1986 Oct 17;873(3):340-9. doi: 10.1016/0167-4838(86)90082-8.
Murine monoclonal antibodies to the extracellular hemoglobin of Lumbricus terrestris were prepared by a modification of the method of Kohler and Milstein. 224 hybridomas were found to produce antibodies which bound to the hemoglobin; they were tested for binding to the four subunits of the hemoglobin: M (chain I, 16 kDa), D1 (chain V, 31 kDa), D2 (chain VI, 37 kDa) and T (50 kDa), a disulfide-bonded trimer of chains II, III and IV, each of about 17 kDa. 150 hybridomas bound to all four subunits and 40 hybridomas bound to various combinations of subunits. The remaining 34 hybridomas combined only with the hemoglobin. The twelve hybridomas obtained after subculturing and cloning were tested for their binding to the two fractions II and III, consisting of subunits D1 + D2 + T and M, respectively, obtained by dissociation at pH 9.5 and at pH 4.0 and to the reassociated whole molecules, obtained subsequent to return to neutral pH. Eight hybridomas which combined only with the hemoglobin also combined with all the reassociated molecules but not with any of the fractions: these monoclonal antibodies probably recognize conformation-dependent antigenic sites that are present only in the hexagonal bilayer structure characteristic of the native and reassociated hemoglobin molecules. Of the remaining four hybridomas, two bound to subunit T and two combined with subunits T and D2; they also combined with the reassociated molecules and with the fractions II. In addition, the hybridomas did not bind to the hemoglobins of Tubifex, Limnodrilus, Arenicola, Tylorrhynchus and Macrobdella or to the chlorocruorins of Myxicola and Eudistylia.
通过对科勒和米尔斯坦方法的改进，制备了针对蚯蚓细胞外血红蛋白的鼠单克隆抗体。发现224个杂交瘤产生与血红蛋白结合的抗体；对它们与血红蛋白的四个亚基的结合情况进行了测试：M（链I，16 kDa）、D1（链V，31 kDa）、D2（链VI，37 kDa）和T（50 kDa），T是由链II、III和IV组成的二硫键连接的三聚体，每条链约17 kDa。150个杂交瘤与所有四个亚基结合，40个杂交瘤与亚基的各种组合结合。其余34个杂交瘤仅与血红蛋白结合。对传代培养和克隆后获得的12个杂交瘤进行了测试，检测它们与分别由pH 9.5和pH 4.0解离得到的、由亚基D1 + D2 + T和M组成的II和III组分的结合情况，以及与恢复到中性pH后重新结合的完整分子的结合情况。8个仅与血红蛋白结合的杂交瘤也与所有重新结合的分子结合，但不与任何组分结合：这些单克隆抗体可能识别仅存在于天然和重新结合的血红蛋白分子特有的六边形双层结构中的构象依赖性抗原位点。在其余4个杂交瘤中，2个与亚基T结合，2个与亚基T和D2结合；它们也与重新结合的分子和II组分结合。此外，这些杂交瘤不与颤蚓、水丝蚓、沙蠋、吻蚓和巨蛭的血红蛋白或黏液虫和优美盘管虫的绿血蛋白结合。
