de Jong J G, Amesz H, Aarsman A J, Lenting H B, van den Bosch H
Eur J Biochem. 1987 Apr 1;164(1):129-35. doi: 10.1111/j.1432-1033.1987.tb11003.x.
The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzyme-linked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A--Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondrial phospholipase A2) antibodies.
大鼠肝线粒体膜相关磷脂酶A2通过AcA 54凝胶过滤和Matrex凝胶蓝A色谱法进行溶解和部分纯化。将大约纯化了2500倍的制剂注射到小鼠体内，在脾细胞与小鼠SP2/0骨髓瘤细胞融合后制备抗磷脂酶A2的单克隆抗体。用部分纯化的磷脂酶A2作为抗原，通过酶联免疫吸附测定法检测杂交瘤上清液中的抗体产生。在涉及与兔抗（小鼠免疫球蛋白）和杂交瘤上清液中的单克隆抗体复合的蛋白A-琼脂糖的特异性免疫沉淀测定中，检测阳性克隆结合磷脂酶A2的能力。鉴定出12个产生结合线粒体磷脂酶A2抗体的克隆。所有这些抗体与从AcA 54和Matrex凝胶蓝A柱洗脱的蛋白组分的结合与这些组分中的磷脂酶A2活性一致。所有单克隆抗体均与大鼠肝细胞质和溶解的大鼠血小板磷脂酶A2表现出交叉反应性。抗（线粒体磷脂酶A2）抗体未识别大鼠和猪胰腺或响尾蛇的细胞外磷脂酶A2。
