EP8.0细菌内毒素检查法中英文对照.doc

EP8.0 2.6.14 细菌内毒素 （中英文 ） 2.6.14. BACTERIAL ENDOTOXINS 细菌内毒素 The test for bacterial endotoxins (BET) is used to detect or quantify endotoxins from gram-negative bacteria using amoebocyte lysate from the horseshoe crab (Limulus polyphenmus or Tachypleus tridentatus). There are 3 techniques for this test: the gel-clot techniques, which is based on gel formation; the turbidimetric technique, based on the development of turbidity after cleavage of an endogenouse substrate; and the chromogenic technique, based on the development of colour after cleavage of a synthetic peptide-chromogen complex. 本法利用鲎试剂（从鲎——美洲鲎或中国鲎——变形细胞溶解物制备而来）检测由革兰氏阴性菌产生的细菌内毒素或对内毒素进行定量。该检查包括三种方法：一为凝胶法，系利用鲎试剂与内毒素产生凝集反应的原理；第二种为浊度法（基于内源性底物断裂后，产生的浊度变化）；最后一种为显色法（得到的肽－呈色基团复合物断裂后，检测反应混合物的色度）。 The following 6 methods are described in the present chapter:这一章阐述了下面6种方法： Method A. Gel-clot method: limit test Method B. Gel-clot method: quantitative test Method C. Turbidimetric kinetic method Method D. Chromogenic kinetic method Method E. Chromogenic end-point method Method F. Turbidimetric end-point method 方法A：凝胶法：限度试验 方法B：凝胶法：定量试验 方法C：动态浊度法 方法D：动态显色法 方法E：终点显色法 方法F：终点浊度法 Proceed by any of the 6 methods for the test. In the event of doubt or dispute, the final decision is made based upon method A unless otherwise indicated in the monograph. 检测时，可用6种方法的任一种进行试验。当测定结果可疑或有争议时，除非另有规定，以专论中的方法A的测定结果为准。 The test is carried out in a manner that avoids endotoxin contamination. 试验操作过程应防止内毒素的污染。 1. APPARATUS仪器 Depyrogenate all glassware and other heat-stable apparatus in a hot-air oven using a validated process. A commonly used minimum time and temperature is 30 minutes at 25?C. If employing plastic apparatus, such as microtitre plates and pipette tips for automatic pipetters, use apparatus shown to be free of detectable endotoxin and which does not interfere in the test. 所有的玻璃器皿及由其他耐热材料制成的器皿需用已验证的工艺在热烘箱内进行去热原处理。去热原时，常用的最小时间和温度设置分别为30分钟和250℃。若使用塑料器械，如微孔板和微量进样器配套的吸头等，它们必须标明无内毒素并确对试验无干扰。 NOTE: in this chapter, the term ‘tube’ includes all types of receptacles, for example microtitre plate wells. 注：这一章中，“管”的意思包括其他任何反应容器，如微孔板中的孔。 2. REAGENTS, TEST SOLUTIONS试剂、试液 (1) Amoeboyte lysate鲎试剂 Amoebocyte lysate is a lyophilized product obtained from amoebocyte lysate from the horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). This reagent refers only to a product manufactured in accordance with the regulations of the competent authority. 鲎试剂物是从鲎（美洲鲎或中国鲎）的变形细胞产生的低压冻干产物。该试剂仅指在符合有关权威法规的生产条件下生产的鲎试剂产品。 NOTE: amoebocyte lysate reacts with some β-glucans in addition to endotoxins. Amoebocyte lysate preparations which do not reacto with glucans are available; they are prepared by removing from amoebocyte lysate the G factor, which reacts with glucans, or by inhibiting the G factor reactoing system of amoebocyte lysate. These preparations may be used for endotoxin testing in the presence of glucans. 注：除了内毒素外，鲎试剂和β-葡聚糖反应。也存在不与葡聚糖反应的鲎试剂；它们通过从变形细胞溶解物中除去G因子来制备，因为G因子会和葡聚糖反应，或者通过抑制变形细胞溶解物的G因子反应系统来制备。这些制剂可被用于葡聚糖存在情况下的内毒素测试。 (2) Lysate solution鲎试液 Dissolve amoebocyte lysate in water for BET or in a buffer, as recommended by the lysate manufacturer, by gentle stirring. Store the reconstituted lysate, refrigerated or frozen, as indicated by the manufacturer. 通过缓慢搅拌，将鲎试剂溶于水或溶于厂家推荐的缓冲液中来进行细菌内毒素试验（BET）。按照厂商的指示，冷藏或冷冻储存重新鲎试液。 (3) Water for BET (water for bacterial endotoxins test) BET用水 Water for injections R or water produced by other procedures that shows no reaction with the lysate employed at the detection limit of the reagent. BET用水指注射用水R或由其他方法制取的内毒素含量低于所用鲎试剂的检测限的水。 3. PREPARATION OF THE STANDARD ENDOTOXIN STOCK SOLUTION（内毒素储备标准溶液的制备） The standard endotoxin stock solution is prepared from an endotoxin reference standard that has been calibrated against the International Standard, for example endotoxin standard BRP. 用内毒素标准品制备内毒素储备标准溶液；所用的内毒素标准品必须先用国际标准品校准，如内毒素标准BRP。 Endotoxin is expressed in International Units (IU). The equivalence in IU of the International Standard is stated by World Health Organisation. 内毒素以国际单位（IU）表示。IU的换算见国际卫生组织（WHO）公布的国际标准。 NOTE: one International Unit (IU) of endotoxin is equal to one Endotoxin Unit (E.U.). 注：一国际单位（IU）内毒素相当于一个内毒素单位（E.U.）。 Follow the specifications in the package leaflet and on the lable for preparation and storage of the standard endotoxin stock solution. 根据包装说明书上的标准和内毒素储备标准溶液的标签上关于制备和贮存的说明。 4. PREPARATION OF THE STANDARD ENDOTOXIN SOLUTIONS（ 内毒素标准溶液的制备） After vigorously mixing the standard endotoxin stock solution, prepare appropriate serial dilutions of this solution using water for BET. 充分混合内毒素储备标准溶液后，用细菌内毒素试验检查用水（BET用水）稀释，制成适当的系列稀释液，即得BET用内毒素标准溶液。 Use the solution as soon as possible to avoid loss of activity by adsorption. 得到的稀释液应尽快使用，以免因吸附而导致活性损失。 5. PREPARATION OF THE TEST SOLUTIONS供试品溶液的制备 Prepare the test solutions by dissolving or diluting active substances or medicinal products using water for BET. Some substances or preparations may be more appropriately dissolved or diluted in ot