Young S G, Witztum J L, Casal D C, Curtiss L K, Bernstein S
Arteriosclerosis. 1986 Mar-Apr;6(2):178-88. doi: 10.1161/01.atv.6.2.178.
The fact that low density lipoprotein (LDL) from multiple animal species binds to the human LDL receptor suggested that the LDL-receptor binding domain of apoprotein (apo)B must be evolutionarily conserved. To determine if a common receptor domain epitope existed on apo B, we generated a monoclonal antibody that was specific for the LDL-receptor domain of apo B. This was accomplished by using a screening procedure that selected for a hybridoma supernatant that could block specific cellular uptake and degradation of LDL. Western blots showed that this antibody, termed MB47, was specific for apo B-100. Fluid phase assays indicated a high binding affinity (Ka = 4 X 10(9) M-1) and demonstrated that all human LDL particles expressed the MB47 epitope. Scatchard analysis indicated that a maximum of one MB47 molecule bound to each LDL particle. In solid phase assays, antibody MB47 bound to plasma or LDL of multiple mammalian species, including guinea pig, rabbit, pig, dog, cat, seal, whale, bear, and lion, but it did not bind to mouse or rat LDL. In contrast, a rabbit antiserum to LDL and two other anti-apo B monoclonal antibodies, MB3 and MB19, which do not bind to the receptor domain, were specific only for human LDL. LDL from multiple species, including mouse LDL, competed effectively with 125I-human LDL for binding to human fibroblasts. MB47 effectively inhibited uptake and degradation of labeled human, guinea pig, and rabbit LDL by both human and guinea pig fibroblasts. We conclude that antibody MB47 binds to a single receptor domain on LDL and identifies a vital region conserved through mammalian evolution.
多种动物物种的低密度脂蛋白（LDL）能与人LDL受体结合，这一事实表明载脂蛋白（apo）B的LDL受体结合域在进化上一定是保守的。为了确定apo B上是否存在共同的受体结构域表位，我们制备了一种对apo B的LDL受体结构域具有特异性的单克隆抗体。这是通过一种筛选程序实现的，该程序选择能阻断LDL特异性细胞摄取和降解的杂交瘤上清液。蛋白质印迹法显示，这种名为MB47的抗体对apo B - 100具有特异性。液相分析表明其具有高结合亲和力（Ka = 4×10⁹ M⁻¹），并证明所有人LDL颗粒都表达MB47表位。Scatchard分析表明每个LDL颗粒最多结合一个MB47分子。在固相分析中，抗体MB47能与多种哺乳动物物种的血浆或LDL结合，包括豚鼠、兔子、猪、狗、猫、海豹、鲸鱼、熊和狮子，但不与小鼠或大鼠的LDL结合。相比之下，一种针对LDL的兔抗血清以及另外两种不与受体结构域结合的抗apo B单克隆抗体MB3和MB19仅对人LDL具有特异性。包括小鼠LDL在内的多种物种的LDL能有效地与¹²⁵I - 人LDL竞争结合人成纤维细胞。MB47能有效抑制人和豚鼠成纤维细胞对标记的人、豚鼠和兔子LDL的摄取和降解。我们得出结论，抗体MB47与LDL上的单个受体结构域结合，并识别出一个在哺乳动物进化过程中保守的重要区域。
