Development Of New Assays For Lipoprotein Testing

脂蛋白分数分析有助于估计冠状动脉疾病的风险和评估降胆固醇治疗的有效性。本研究的目的是提高当前脂蛋白检测的分析性能，并有可能开发新的脂蛋白检测方法，提高诊断准确性。在过去的一年里，我们已经开发了几种替代的洗涤剂增溶程序，使我们之前开发的顺序脂蛋白测试适应于常规临床使用的其他脂蛋白均质分析测试。序列脂蛋白测试可以用自动化仪器在一个反应管中测量所有主要的血清脂质和脂蛋白组分（高密度脂蛋白胆固醇、总胆固醇、甘油三酯和计算的低密度脂蛋白胆固醇），从而大大简化了脂蛋白组分分析的实验室程序。我们还研究了利用MALDI-TOF质谱法对载脂蛋白（脂蛋白颗粒上的主要蛋白质）的免疫测定捕获技术进行表征。使用该技术，我们能够从血清中检测和表征载脂蛋白A-I和载脂蛋白E的分子形式，这可能是诊断标志物。例如，载脂蛋白E的某些分子形式已被证明与阿尔茨海默病的发展有关。但是目前用于鉴定载脂蛋白E的不同分子表型的测试是非常劳动密集型的，而且执行起来很昂贵。在未来，我们计划开发一种检测方法，通过免疫测定捕获和MALDI-TOF串联质谱法进行载脂蛋白E表型分析，这将允许根据分子量检测和区分各种形式的载脂蛋白E。我们还计划研究几种沉淀和过滤方法，以开发一种可由常规临床实验室进行的测试，用于测量β - HDL，一种小尺寸HDL颗粒，最近被证明比大尺寸HDL颗粒更好地预测冠状动脉疾病。

Lipoprotein fraction analysis is useful for estimating the risk for coronary artery disease and for assessing the effectiveness of cholesterol lowering therapy. The objective of this study is to improve the analytical performance of current lipoprotein assays and to potentially develop new lipoprotein tests with improved diagnostic accuracy. In the past year, we have developed several alternative detergent solubilization procedures for adapting our previously developed sequential lipoprotein test to other lipoprotein homogenous assay tests that are in routine clinical use. The sequential lipoprotein test makes it possible to measure in a single reaction tube with an automated instrument all the major serum lipid and lipoprotein fractions (HDL-cholesterol, total cholesterol, triglyceride, and calculated LDL-cholesterol), thus significantly simplifying the laboratory procedure for lipoprotein fraction analysis. We also investigated the use of an immunoassay capture technique for the characterization of apolipoproteins, the major proteins on lipoprotein particles, by MALDI-TOF mass spectrometry. Using this technique, we were able to detect and characterize from serum the molecular forms for apolipoprotein A-I and apolipoprotein E, which are possible diagnostic markers. For example, some molecular forms of apolipoprotein E have been shown to be associated with the development of Alzheimer?s disease, but the currently used tests for identifying the different molecular phenotypes of apolipoprotein E are very labor intensive and expensive to perform. In the future, we plan to develop an assay for performing apolipoprotein E phenotyping by immunoassay capture followed by MALDI-TOF tandem mass spectrometry, which will allow the detection and differentiation of the various forms of apolipoprotein E by their molecular weight. We also plan to investigate several precipitation and filtration methods for developing a test that can be performed by routine clinical laboratories for measuring pre-beta HDL, a small size HDL particle that has recently been shown to be a better negative predictor for coronary artery disease than large size HDL particles.