Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100.
Evaluation of a monoclonal antibody-based enzyme-linked immunosorbent assay as a candidate reference method for the measurement of apolipoprotein B-100.
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发表时间：
1989-09
影响因子：
6.5
通讯作者：
J. J. Albers-J.;M. Lodge;Linda K. Curtiss
中科院分区：
文献类型：
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作者：
J. J. Albers-J.;M. Lodge;Linda K. Curtiss
已开发了一种基于单克隆抗体的载脂蛋白（apo）B-100直接结合酶联免疫吸附试验（ELISA），用作参考方法。该测定使用两种充分表征的单克隆抗体MB 24和MB 47。MB 47识别apoB低密度脂蛋白（LDL）受体结合域的表位，对apoB-100具有特异性，作为捕获抗体与微量滴定板结合。MB 24结合apoB-100氨基末端一半的表位并鉴定apoB-100和apoB-48，与辣根过氧化物酶缀合并用作指示抗体。用LDL（d 1.030-1.050 g/ml）校准测定法，并通过十二烷基硫酸钠（SDS）Lowry程序测定LDL蛋白。测定的工作范围为0.25-1.25 μ g/ml。发现全血浆的最佳稀释度为1：2000。在测定中，MB 47结合所有低密度脂蛋白中约97%的apoB，并且在大多数极低密度脂蛋白制剂中大于90%的apoB。当在参考ELISA中测试时，来自家族性混合型高脂血症（FCHL）受试者的小密度LDL和来自家族性高胆固醇血症（FH）受试者的大浮力LDL表现出与来自健康血脂正常受试者的LDL相似的结合特性。批内和批间变异系数平均分别为2.5%和6.0%。血浆B-100水平不受冻融或在4 ℃下储存长达3周或在-70 ℃下储存长达11个月的影响。在参考ELISA和测量总apo B的多克隆RIA之间获得了极好的一致性（r = 0.93，n = 105，平均ELISA B-100值= 100 mg/dl，平均RIA值= 101 mg/dl，Sy = 9.6）。在血浆甘油三酯水平低于600 mg/dl的大多数样本中，用细菌脂肪酶预处理的样本的参考ELISA B-100值未显著增加。为了帮助减少apoB测量的大的实验室差异性，我们建议使用该候选参考直接结合ELISA将apoB目标值分配给apoB参考池。
A monoclonal antibody-based direct binding enzyme-linked immunosorbent assay (ELISA) for apoprotein (apo) B-100 has been developed for use as a reference method. The assay uses the two well-characterized monoclonal antibodies, MB24 and MB47. MB47, which recognizes an epitope at the low density lipoprotein (LDL) receptor-binding domain of apoB and is specific for apoB-100, is bound to the microtiter plate as the capture antibody. MB24, which binds an epitope in the amino terminal half of the apoB-100 and identifies both apoB-100 and apoB-48, is conjugated to horseradish peroxidase and is utilized as the indicating antibody. The assay was calibrated with LDL (d 1.030-1.050 g/ml) and the LDL protein was determined by a sodium dodecyl sulfate (SDS) Lowry procedure. The working range of the assay is 0.25-1.25 micrograms/ml. Optimal dilution of whole plasma was found to be 1:2000. In the assay, MB47 bound approximately 97% of the apoB in all low density lipoprotein, and greater than 90% of the apoB in the majority of very low density lipoprotein preparations. Small dense LDL from subjects with familial combined hyperlipidemia (FCHL) and large bouyant LDL from subjects with familial hypercholesterolemia (FH) exhibited binding properties similar to LDL from healthy normolipidemic subjects when tested in the reference ELISA. The intra- and interassay coefficients of variation averaged 2.5% and 6.0%, respectively. Plasma B-100 levels were not influenced by freezing and thawing or storage at 4 degrees C for up to 3 weeks or storage at -70 degrees C for up to 11 months. Excellent agreement was obtained between the reference ELISA and a polyclonal RIA which measures total apoB (r = 0.93, n = 105, mean ELISA B-100 value = 100 mg/dl, mean RIA value = 101 mg/dl, Sy = 9.6). Reference ELISA B-100 values of samples pretreated with bacterial lipase were not significantly increased in most samples with plasma triglyceride levels below 600 mg/dl. To help reduce the large among-laboratories variability of apoB measurements, we recommend that this candidate reference direct binding ELISA be used to assign apoB target values to apoB reference pools.