Tatzber F, Wonisch W, Schmidl E, Esterbauer H
Institute of Biochemistry, University of Graz, Austria.
Biofactors. 1997;6(2):125-30. doi: 10.1002/biof.5520060205.
It is generally accepted, that lipid peroxidation plays a pathogenic role in atherosclerosis. Furthermore, recent studies indicate that antibodies directed against oxidative modifications of Low Density Lipoprotein (oLAb) contribute to atherosclerotic processes and may have some function in other disorders. These antibodies have been determined predominantly in humans, because assays for oLAb measurement use species specific anti IgG conjugates. From such assay designs it is not possible to get directly comparable data from various animal species. Main advantages of comparable data between animal species are that results of animal experiments can be interpreted using human calibrators and that results of immunisations and production of monoclonal antibodies are directly comparable not only within, but also between animal species. The aim of this study was to find a modification for ELISAs for oLAb determination, which allows to measure sera of various animal species simultaneously. Microtitration plates were coated with oxidised LDL and blocked with bovine serum albumine. Human and animal sera were then pipetted into the plate in logarithmic serial dilutions and incubated for 2 h at 37 degrees C. After washing, a protein A horse-radish peroxidase conjugate (Biomakor, Israel) was added to each well in a dilution of 1:20,000. The incubation conditions had to be optimized to achieve reliable results. After another washing step, the assay was developed with TMB. Absorptions were read at 450 nm in a microplate photometer. Following the manufacturers incubation instructions, which recommended a duration of 1 h at room temperature, the system did not work optimally. No binding of protein A to IgG molecules bound to oxidised LDL could be observed, if the system was incubated at 37 degrees C. In our hands, best results were achieved for several animal species, if the conjugate was incubated for two hours at 2-4 degrees C in a refrigerator. Under these conditions, assay sensitivity was the same as in the standard method, which uses anti-species IgG conjugates. The protein A modification of oLAb allows direct reading of animal oLAb titres from human calibrators. With this method, results of animal experiments can be interpreted on the basis of the situation in humans. Preliminary results obtained show that immunisation experiments with oxidised LDL give serum titres in animals, which are in the same order of magnitude as human sera with high oLAb concentrations. The results of this study, in accordance with findings of other authors, give further indications that atherosclerotic processes are influenced by the specific immune system.
一般认为，脂质过氧化在动脉粥样硬化中起致病作用。此外，最近的研究表明，针对低密度脂蛋白氧化修饰的抗体（oLAb）有助于动脉粥样硬化进程，并且可能在其他疾病中发挥某些作用。这些抗体主要在人类中检测，因为用于测量oLAb的检测方法使用物种特异性抗IgG缀合物。从这样的检测设计中，无法直接获得来自各种动物物种的可比数据。动物物种间可比数据的主要优点是，动物实验结果可以使用人类校准物进行解释，并且免疫和单克隆抗体制备的结果不仅在动物物种内，而且在不同动物物种间都可以直接比较。本研究的目的是找到一种用于测定oLAb的酶联免疫吸附测定（ELISA）的改进方法，该方法允许同时检测各种动物物种的血清。微量滴定板用氧化型低密度脂蛋白包被，并用牛血清白蛋白封闭。然后将人和动物血清以对数系列稀释液加入板中，并在37℃孵育2小时。洗涤后，将蛋白A辣根过氧化物酶缀合物（以色列Biomakor公司）以1:20,000的稀释度加入每个孔中。必须优化孵育条件以获得可靠的结果。在另一个洗涤步骤后，用四甲基联苯胺（TMB）显色。在酶标仪中于450nm处读取吸光度。按照制造商的孵育说明，建议在室温下孵育1小时，但该系统不能最佳运行。如果在37℃孵育该系统，则未观察到蛋白A与结合到氧化型低密度脂蛋白上的IgG分子结合。在我们的实验中，如果将缀合物在2-4℃的冰箱中孵育两小时，对于几种动物物种可获得最佳结果。在这些条件下，检测灵敏度与使用抗物种IgG缀合物的标准方法相同。oLAb的蛋白A修饰允许直接根据人类校准物读取动物oLAb滴度。用这种方法，动物实验结果可以根据人类情况进行解释。获得的初步结果表明，用氧化型低密度脂蛋白进行免疫实验在动物中产生的血清滴度与高oLAb浓度的人类血清处于相同的数量级。本研究的结果与其他作者的发现一致，进一步表明动脉粥样硬化进程受特异性免疫系统的影响。
