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1、 细菌内毒素检测细菌内毒素检测 Portions of this general chapter have been harmonized with the corresponding texts of the European Pharmacopoeia and/or the Japanese Pharmacopoeia. Those portions that are not harmonized are marked with symbols to specify this fact. 本文中部分内容与欧洲药典和/或日本药典的相应的内容是一致的，那些不一致的内容则用符号（） 标明 The
2、 Bacterial Endotoxins Test (BET) is a test to detect or quantify endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). 细菌内毒素测试(BET)是检测或定量革兰氏阴性细菌内毒素的试验，它使用从鲎（美洲鲎或东方鲎）的 循环阿米巴细胞的抽提液中制备的鲎试剂。 There are three techniques for t
3、his test: the gel-clot technique, which is based on gel formation; the turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate; and the chromogenic technique, based on the development of color after cleavage of a synthetic peptide-chromogen complex. P
4、roceed by any of the three techniques for the test. In the event of doubt or dispute, the final decision is made based upon the gel-clot limit test 2s(USP 35) unless otherwise indicated in the monograph for the product being tested. The test is carried out in a manner that avoids endotoxin contamina
5、tion. 细菌内毒素试验有三种技术：基于凝胶形成的凝胶法；基于一内源性基质断裂后的浊度变化；和显色基质法 基于一个带有生色团的合成肽断裂后颜色的变化。除非文中另有说明，否则可以用三种技术中的任何一 种进行测试。若检测结果存在争议，除非文中另有说明，以凝胶法结果为准。该试验以一种避免内毒素污染 的方式进行。 APPARATUS 仪器仪器设备设备 Depyrogenate all glassware and other heat-stable materials in a hot air oven using a validated process. 1 A commonly used mini
6、mum time and temperature is 30 min at 250 . If employing plastic apparatus, such as microplates and pipet tips for automatic pipetters, use apparatus that is shown to be free of detectable endotoxin and does not interfere in the test. NOTEIn this chapter, the term “tube” includes any other receptacl
7、e such as a microtiter well. 所有玻璃用具和耐热材料应置于热空气烤箱中有效除热原。 通常使用的最少时间和最低温度为 30 分钟， 250。 如果使用塑料用具，如微孔板和自动加样器配套的吸头之类的，应确认其不含可测内毒素并不会干扰试验。 注：在这一章中， “管”这个词泛指其它任何容器，如微量孔板的孔 REAGENTS AND TEST SOLUTIONS 试剂和测试方法试剂和测试方法 Amoebocyte LysateA lyophilized product obtained from the lysate of amoebocytes (white blood c
8、ells) from the horseshoe crab (Limulus polyphemus or Tachypleus Solutridentatus). This reagent refers only to a product manufactured in accordance with the regulations of the competent authority. NOTEAmoebocyte Lysate reacts to some -glucans in addition to endotoxins. Amoebocyte Lysate preparations
9、that do not react to glucans are available: they are prepared by removing the G factor reacting to glucans from Amoebocyte Lysate or by inhibiting the G factor reacting system of Amoebocyte Lysate and may be used for endotoxin testing in the presence of glucans. 鲎试剂是一种冻干的产物，它从鲎 （美洲鲎或东方鲎）的阿米巴细胞（白血细胞）
10、的溶解产物中获得。本试 剂仅指根据主管当局的规定而生产的产品。注：除了内毒素，鲎试剂也与某些 -葡聚糖反应。一些经过处 理而制备的鲎试剂不会与 -葡聚糖反应， 它们是通过除去阿米巴细胞产生的 G 因子或抑制阿米巴细胞反应系 统 G 因子的反应机制实现的，对于含有葡聚糖的样品的检测应用此类试剂。 Water for Bacterial Endotoxins Test (BET)Use Water for Injection or water produced by other procedures that shows no reaction with the lysate employed,
11、at the detection limit of the reagent. 用于细菌内毒素测试的水(注):在试剂的检测范围内，使用注射用水或不含与鲎试剂反应的物质的水。 Lysate TSDissolve Amoebocyte Lysate in Water for BET, or in a buffer recommended by the lysate manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen, according to the specificatio
12、ns of the manufacturer. 将鲎试剂溶解于水中，或在制造商推荐的缓冲液中温和搅拌，然后根据说明书将重组的鲎试剂冷藏或冷冻。 1For a validity test of the procedure for inactivating endotoxins, see Dry-Heat Sterilization under Sterilization and Sterility Assurance of Compendial Articles . Use Lysate TS having a sensitivity of not less than 0.15 Endotoxi
13、n Unit per mL. 为使内毒素进行有效的检验，选择干热灭菌，并保证遵照条例的规定。使用的是一种敏感性每毫升不少于0.15个内毒素 单位的鲎试剂。 PREPARATION OF SOLUTIONS 准备的解决方案准备的解决方案 Standard Endotoxin Stock SolutionA Standard Endotoxin Stock Solution is prepared from a USP Endotoxin Reference Standard that has been calibrated to the current WHO International Sta
14、ndard for Endotoxin. Follow the specifications in the package leaflet and on the label for preparation and storage of the Standard Endotoxin Stock Solution. Endotoxin is expressed in Endotoxin Units (EU). NOTEOne USP Endotoxin Unit (EU) is equal to one International Unit (IU) of endotoxin. 标准内毒素溶液是按
15、 USP 内毒素参考标准制备的，该标准已相当于目前国际内毒素的标准。按照标准内毒素 溶液说明书和和储存标签的说明，内毒素的量用 USP 内毒素单位（USP-EU）表示。注 1:一种 USP 内毒素单位 (EU)等于一国际单位(国际单位)内毒素。注：1 USP-EU 内毒素等于国际单位 1 IU 的内毒素 Standard Endotoxin SolutionsAfter mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial dilutions of Standard Endotox
16、in Solution, using Water for BET. Use dilutions as soon as possible to avoid loss of activity by adsorption. 标准内毒素溶液： 在将标准内毒素溶液混合后， 将标准内毒素溶液进行适当的连续稀释。 尽可能使用稀释剂， 以避免因吸附而失去活性。 Sample SolutionsPrepare the Sample Solutions by dissolving or diluting drugs 2S(USP35)using Water for BET. Some substances or preparations may be more appropriately dissolved, or diluted 2s(usp35) in other aqueous solutions. If necessary, adjust the pH of the solution to be examined (or
