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

在建立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分析方法之前，研究了血清样品的前处理方法，建立了大鼠载脂蛋白E和载脂蛋白A-I的酶联免疫吸附分析方法。1)建立了夹心法测定大鼠载脂蛋白E。从血清脂蛋白中提纯载脂蛋白E，制备兔抗血清。将稀释后的样品和标准品加入包被纯化的免疫球蛋白抗体的微量板孔中孵育。洗涤后，将纯化的Fab‘-辣根过氧化物酶结合物加入每口井并孵化。用比色法测定结合酶的含量。样品和标准品用6M盐酸胍处理，以最大限度地提高载脂蛋白E的抗原性，灵敏度约为1pg，工作范围为0.1~1.0 ng。平均批内和批间变异系数分别为1.8%和4.1%。青年大鼠(8~12周龄，n=9)和老年大鼠(36~40周龄，n=16)的血清载脂蛋白E浓度分别为21.2±lt；+-gt；2.1和61.3；+-gt；17.0 mg/dl(平均值+lt；+-gt；SD)。凝胶过滤分析表明，禁食大鼠血清中的载脂蛋白E大多与较大的高密度脂蛋白颗粒有关，有一小部分载脂蛋白E以游离形式存在。2)建立了大鼠载脂蛋白A-I的夹心酶联免疫吸附试验。用6M盐酸胍处理血清样本，可最大限度地暴露载脂蛋白A-I表位。灵敏度达到1pg，工作范围为0.1~1.0 ng。平均批内和批间变异系数分别为2.8%和4.1%。老年(36-40周龄)大鼠血清载脂蛋白A-I浓度(62.3；+-gt；8.6 mg/dl，均值为8.6 mg/dl)显著高于青年(8-12周龄)大鼠(55.1；+-gt；4.3 mg/dl，n=16)。

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