Development of a therapeutic anti-marinobufagenin antibody

虽然先兆子痫是母亲和胎儿死亡的主要原因，但其发病机制尚未完全了解。内源性洋地黄样强心类固醇（CTS）与先兆子痫的病理生理学有关，如临床观察所示，Digibind（一种结合CTS的地高辛抗体）降低先兆子痫患者的血压。最近，我们报道了严重PE患者血浆中蟾毒配基（MBG）（一种蟾毒类血管收缩剂CTS）的水平增加了4倍。在本研究中，我们比较了正常和先兆子痫胎盘中MBG的水平，并测试了针对MBG和哇巴因的抗体与通过高效液相色谱法（HPLC）从先兆子痫胎盘纯化的材料的相互作用。MBG的水平，但不是内源性哇巴因，表现出四倍的升高先兆子痫胎盘。内源性胎盘MBG样免疫反应物质从反相HPLC柱的洗脱时间与真实的MBG相同。基于Digibind的免疫测定未检测到与含有哇巴因样免疫反应性物质的HPLC的交叉反应性，但与具有与MBG和其他蟾蜍二烯内酯的保留时间相似的保留时间的HPLC级分发生交叉反应。2008年，我们还比较了Digibind和单克隆抗MBG抗体在两种PE大鼠模型中的体内疗效，即在妊娠第14-20天期间通过补充NaCl导致高血压的正常和糖尿病妊娠大鼠中。在这两种模型中，MBG水平显著升高，抗MBG抗体在降低血压和逆转MBG靶酶Na/K-ATP酶抑制方面超过Digibind。我们的研究结果表明，无论是全身和胎盘MBG水平显着升高先兆子痫，并建议MBG是一个潜在的目标免疫中和这种综合征患者。

Although preeclampsia is a major cause of maternal and fetal mortality, its pathogenesis is not fully understood. Endogenous digitalis-like cardiotonic steroids (CTS) are implicated in the pathophysiology of preeclampsia, as illustrated by clinical observations that Digibind, a digoxin antibody which binds CTS, lowers blood pressure in patients with preeclampsia. Recently we reported that plasma levels of marinobufagenin (MBG), a bufadienolide vasoconstrictor CTS, are increased four-fold in patients with severe PE. In the present study, we compared levels of MBG in normal and preeclamptic placentae, and tested antibodies against MBG and ouabain for their interaction with the material purified from preeclamptic placentae via high-performance liquid chromatography (HPLC). Levels of MBG, but not that of endogenous ouabain, exhibited a four-fold elevation in preeclamptic placentae. The elution time of endogenous placental MBG-like immunoreactive material from reverse-phase HPLC column was identical to that of authentic MBG. Immunoassay based on Digibind did not detect cross-reactivity with HPLC containing ouabain-like immunoreactive material, but cross-reacted with HPLC fractions having retention time similar to that of MBG and other bufadienolides. In 2008 we also compared the in vivo efficacy of Digibind and monoclonal anti-MBG antibodies in two rat models of PE, in normal and diabetic pregnant rats rendered hypertensive by NaCl supplementation during days 14-20 of gestation. In both models, levels of MBG were substantially elevated, and anti-MBG antibody exceeded Digibind in its capacity to lower blood pressure and to reverse inhibition of Na/K-ATPase, a target enzyme for MBG. Our results demonstrate that both systemic and placental levels of MBG are significantly elevated in preeclampsia, and suggest that MBG is a potential target for immunoneutralization in patients with this syndrome.