Enzyme-Linked Immunosorbent Assay of Serum Low Density Lipoproteins Modified by Lipid Peroxidation Products

在建立由脂蛋白中脂质过氧化物降解产物修饰的低密度脂蛋白的酶联免疫吸附法之前，研究了血清样品的预处理方法，并建立了大鼠载脂蛋白E和A-I的酶联免疫吸附法。1)建立了大鼠载脂蛋白E （apo E）夹心酶联免疫吸附法。从兔血清脂蛋白中纯化载脂蛋白E，制备抗血清。将稀释后的样品和标准品加入预先包被纯化IgG的微滴板孔中孵育。洗涤后，将纯化的Fab′-辣根过氧化物酶偶联物加入每孔孵育。结合酶用比色法测定。样品和标准品用6 M胍- hci预处理，最大限度地提高载脂蛋白E的抗原反应，灵敏度在1 pg左右，工作范围为0.1 ~ 1.0 ng。平均组内变异系数为1.8，组间变异系数为4.1%。幼龄大鼠（8-12周龄，n = 9）和老年大鼠（36-40周龄，n = 16）血清载脂蛋白E浓度分别为21.2 <正负> 2.1和61.3 <正负> 17.0 mg/dl（平均<正负> SD）。凝胶过滤发现，禁食大鼠血清中的大部分载脂蛋白E与较大的HDL颗粒有关，并且在free2中存在一小部分载脂蛋白E。2)同样开发了大鼠载脂蛋白a - i（载脂蛋白a - i）的夹心酶联免疫吸附法。用6 M胍-盐酸处理血清样品，最大限度地暴露载脂蛋白A-I的表位。灵敏度可达1 pg，工作范围为0.1 ~ 1.0 ng。平均组内变异系数为2.8，组间变异系数为4.1%。老年（36-40周龄）大鼠血清载脂蛋白A-I浓度（62.3 <正负> 8.6 mg/dl，即<正负> SD, n = 16）显著高于幼年（8-12周龄）大鼠（55.1 <正负> 4.3 mg/dl, n = 0.05）

Before developing an enzyme-linked immunosorbent assay of low density lipoproteins modified by degradation products of lipid peroxides in lipoproteins, methods for pretreatment of serum samples were studied and enzyme-linked immunosorbent assays of rat apolipoproteins E and A-I were established.1) A sandwich enzyme-linked immunosorbent assay of rat apolipoprotein E (apo E) was developed. Apo E was purified from serum lipoproteins and antiserum was raised in rabbits. Diluted samples and standards were added into the wells of microtiter plates precoated with purified IgG and incubated. After washing, purified Fab'-horseradish peroxidase conjugate was added to each well and incubated. The bound enzyme was assayed by a colorimetric method. Samples and standards were pretreated with 6 M guanidine-HCI to maximize the antigenic response of apo E. The sensitivity lies around 1 pg apo E, and the working range was 0.1 to 1.0 ng. The mean intra- and interassay coefficients of variation were 1.8 and 4.1%, respectively. Serum apo E concentrations were 21.2 <plus-minus> 2.1 and 61.3 <plus-minus> 17.0 mg/dl (mean <plus-minus> SD) for young (8-12 weeks old, n = 9) and old (36-40 weeks old, n = 16) rats, respectively. As determined by gel filtrations, most of the apo E in fasted rat serum was associated with larger HDL particles and a small portion of apo E was present in a free2) A sandwich enzyme-linked immunosorbent assay for rat apolipoprotein A-I (apo A-I) was also similarly developed. Treatment of serum samples with 6 M guanidine-HCl gave maximum exposure of the epitopes of apo A-I. The sensitivity reached to 1 pg, and the working range was 0.1 to 1.0 ng. The mean intra- and interassay coefficients of variation were 2.8 and 4.1%, respectively. Serum apo A-I concentrations in old (36-40 weeks old) rats (62.3 <plus-minus> 8.6 mg/dl, means <plus-minus> SD, n = 16) were significantly (p< 0.05) higher than those in young (8-12 weeks old) rats (55.1 <plus-minus> 4.3 mg/dl, n