Okada N, Tanaka H, Takizawa H, Okada H
Department of Molecular Biology, Nagoya City University School of Medicine, Japan.
J Immunol. 1995 Jun 1;154(11):6103-11.
MCA44 is a mAb with the capacity to sensitize neuraminidase-treated guinea pig E for hemolysis by homologous guinea pig C, and the Fab fragments of this mAb could also sensitize guinea pig E interfering with the function of a membrane inhibitor of C on guinea pig E. Using an immunosorbent column to which MCA44 was coupled, the antigenic molecule termed 44Ag was purified from the glycoprotein fraction extracted from E membranes. C intermediate sheep E treated with guinea pig C1 and C4 after sensitization with Ab (EAC14b cells) lost the ability to generate C3 convertase with C2 after incubation with 44Ag. Treatment of guinea pig E and PBL with phosphatidyl-inositol specific phospholipase C (PIPLC) partially removed 44Ag, as determined by flow cytometric analysis after immunofluorescence staining with MCA44. However, 125I-labeled 44Ag adsorbed to human E was efficiently removed by PIPLC treatment with a slight reduction in M(r). The 44Ag purified on an immunosorbent column showed three bands on SDS-PAGE. However, partial N-terminal amino acid sequences of the 55-kDa, 70-kDa, and 88-kDa bands under nonreducing conditions were identical and the sequence was 55% homologous to the N-terminal sequence of human decay accelerating factor (CD55). Intracutaneous administration of MCA44 or its F(ab')2 fragment resulted in increased capillary permeability, even after 3 days, as determined by the appearance of Evans blue spots after i.v. administration of the dye. Because control Abs including anti-class I-MHC did not cause such increased capillary permeability, the increase in permeability caused by MCA44 was likely induced by blocking the function of 44Ag in vivo, indicating a crucial role for these molecules in preventing over-activation of C at the site.
MCA44是一种单克隆抗体，能够使经神经氨酸酶处理的豚鼠红细胞E对同种豚鼠补体C敏感，该单克隆抗体的Fab片段也能使豚鼠红细胞E敏感，干扰补体C在豚鼠红细胞E上的膜抑制剂的功能。使用偶联有MCA44的免疫吸附柱，从红细胞E膜提取的糖蛋白组分中纯化出称为44Ag的抗原分子。用抗体致敏后经豚鼠C1和C4处理的C中间型绵羊红细胞E（EAC14b细胞），在与44Ag孵育后，与C2一起失去了产生C3转化酶的能力。用磷脂酰肌醇特异性磷脂酶C（PIPLC）处理豚鼠红细胞E和外周血淋巴细胞，通过用MCA44进行免疫荧光染色后的流式细胞术分析确定，部分去除了44Ag。然而，用PIPLC处理能有效去除吸附在人红细胞E上的125I标记的44Ag，其相对分子质量略有降低。在免疫吸附柱上纯化的44Ag在SDS-PAGE上显示出三条带。然而，在非还原条件下，55-kDa、70-kDa和88-kDa条带的部分N端氨基酸序列相同，且该序列与人类衰变加速因子（CD55）的N端序列有55%的同源性。皮内注射MCA44或其F(ab')2片段会导致毛细血管通透性增加，即使在3天后也是如此，这是通过静脉注射染料后出现伊文思蓝斑点来确定的。因为包括抗I类主要组织相容性复合体在内的对照抗体不会引起这种毛细血管通透性增加，所以MCA44引起的通透性增加可能是由于在体内阻断了44Ag的功能，这表明这些分子在防止补体C在该部位过度激活中起关键作用。
