ELISA测定培养细胞中胞液型磷脂酶A_2的含量

Quantitation of the cytosolic phospholipase A_2 in cultured cells by ELISA

目的建立胞液型磷脂酶A2（cytosolicphospholipaseA2,cPLA2）的ELISA。方法以人cPLA2抗体包被微孔板后，加入抗原（标本），再加入辣根过氧化物酶标记的羊抗人IgG作为第二抗体，经邻苯二胺（OPD）显色后测定其490nm处A值，与标准品对比可得cPLA2含量。结果检测灵敏度可达25．0ng／ml，批内和批间变异系数为5．5％和7．8％，cPLA2含量为56和120mg／ml标本的平均回收率均大于80％。结论该方法是一种测定cPLA2含量的简便可靠方法。

Objective To develop an ELISA for the quantitation of cytosolic phospholipase A2(cPLA2) in cultured U937 cells, Methods In this assay, the polyclonal rabbit anti-human cPLA2 antiserum was used as the capture antibody, the horseradish peroxidase conjugated goat-anti - rabbit IgG as the reporter antibody. Purified human cPLA2 dissloved in Tris - HCl buffered saline was used as the standard protein. The concentrations of samples were determined at 490 nm by addition of OPD. Results The detection limit for cPLA2 was 25. 0ng/ml. The coefficients of inter - and extra- assay variation of cPLA2 were 5. 5% and 7. 8% respectively. The recovery of cPLA2 was greater than 80.0% when U937 cell lysate was supplemented exogenously with two different concentrations of cPLA2. Conclusion This ELISA methed for cPLA2 is a simple and precise quantitative assay.