Devaux C A, Nadler P I, Miller G G, Sachs D H
Mol Cell Biochem. 1985 Sep;68(1):31-40. doi: 10.1007/BF00219386.
SJL mice, which are high responders to Staphylococcal nuclease (nuclease), were immunized and used to produce hybridoma cell lines secreting anti-nuclease monoclonal antibodies (mAb). Ten stable clones were derived from a single fusion. Seven of these produced antibodies of the IgG1, kappa isotype and were more precisely characterized for antigenic specificity. Only one hybridoma cell line (54-10-4) produced anti-nuclease antibodies capable of inhibiting enzymatic activity of nuclease. Binding inhibition analyses strongly suggest that the other monoclonal antibodies, which failed to inhibit nuclease activity detect two different antigenic regions, or epitopes, of the molecule: epitope cluster 1 domain is defined by hybridomas 54-2-7, 54-5-2, 54-9-8, and 54-10-8; epitope cluster 2 by 54-5-1 and 54-1-9. Because of its capacity to inhibit nuclease enzymatic activity mAb 54-10-4 was considered specific for a third epitope of the nuclease molecule called epitope 3. Binding studies of these monoclonal antibodies were extended to peptide fragments of the nuclease molecule in order to examine possible cross-reactions with such fragments, as has previously been reported for antibodies purified from polyclonal antisera. Monoclonal antibodies specific for epitope cluster 1 on the native molecule also bound to the fragments 1-126 and 49-149 but failed to bind to fragment 99-149, suggesting that the corresponding epitope(s) is determined by amino acids localized between residues 49 and 99. The epitope clusters 2 and 3 appeared to be expressed only on the native molecule. Monoclonal antibodies of different clusters exhibited very different migration patterns on isoelectric focusing while monoclonal antibodies of the same cluster were indistinguishable, which suggests that they may have originated from the same B cell precursor. Taken together these data suggest that this panel of monoclonal antibodies detects at least three distinct epitopes of the nuclease molecule, one of which could be involved in the determination of the enzymatic site.
SJL小鼠对葡萄球菌核酸酶（核酸酶）反应强烈，对其进行免疫后用于产生分泌抗核酸酶单克隆抗体（mAb）的杂交瘤细胞系。从一次融合中获得了10个稳定的克隆。其中7个产生IgG1、κ同种型的抗体，并对其抗原特异性进行了更精确的表征。只有一个杂交瘤细胞系（54-10-4）产生能够抑制核酸酶酶活性的抗核酸酶抗体。结合抑制分析强烈表明，其他未能抑制核酸酶活性的单克隆抗体检测到该分子的两个不同抗原区域或表位：表位簇1结构域由杂交瘤54-2-7、54-5-2、54-9-8和54-10-8定义；表位簇2由54-5-1和54-1-9定义。由于mAb 54-10-4具有抑制核酸酶酶活性的能力，因此被认为对核酸酶分子的第三个表位具有特异性，该表位称为表位3。这些单克隆抗体的结合研究扩展到核酸酶分子的肽片段，以检查与这些片段可能的交叉反应，正如先前从多克隆抗血清中纯化的抗体所报道的那样。对天然分子上表位簇1具有特异性的单克隆抗体也与片段1-126和49-149结合，但未与片段99-149结合，这表明相应的表位由位于49至99位残基之间的氨基酸决定。表位簇2和3似乎仅在天然分子上表达。不同簇的单克隆抗体在等电聚焦上表现出非常不同的迁移模式，而同一簇的单克隆抗体无法区分，这表明它们可能起源于相同的B细胞前体。综合这些数据表明，这组单克隆抗体检测到核酸酶分子至少三个不同的表位，其中一个可能参与酶位点的确定。
