Tsuji K, Martin P A, Bussey D M
Appl Environ Microbiol. 1984 Sep;48(3):550-5. doi: 10.1128/aem.48.3.550-555.1984.
The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.
采用Zymate机器人系统实现了用于检测内毒素的显色底物鲎试剂（LAL）检测方法的自动化。所开发的软件使机器人能够自动稀释储备参考内毒素标准品（每毫升20,000内毒素单位）以构建五点标准曲线，将样品稀释至适当的检测浓度，并一式两份进行显色底物LAL检测。计算标准曲线的线性度和每个样品中的内毒素浓度，并自动打印结果。在48分钟内，自动化系统一式两份检测三个样品和一个参考标准品以及一个水空白。该检测的灵敏度是孵育时间的函数。分别在孵育10分钟或16分钟时，该检测在每毫升0至1.0内毒素单位或0至0.2内毒素单位的范围内呈线性（r大于0.99）。通过30分钟的孵育，该方法可具有很高的灵敏度，能检测低至每毫升0.003内毒素单位。通过对一种内毒素参考溶液进行八次检测确定的该检测方法的精密度约为6%。为凝胶凝块检测设计的LAL试剂被修改用于显色底物检测。我们描述了显色底物LAL检测的最佳性能条件以及LAL试剂的稳定性。
