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 other aqueous solutions. If necessary, adjust the pH of the test solution (or dilution thereof) so that the pH of the mixture of the lysate and test solution falls within the pH range specified by the lysate manufacturer, usually 6.0 to 8.0. the pH may be adjusted by the use of acid, base or a suitable buffer, as recommended by the lysate manufacturer. Acids and bases may be prepared from concentrates or sol8ids with water for BET in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.
除非另有说明，以BET用水溶解或稀释活性成分或药品来制备供试品溶液。某些成分或药品可能在其它的水溶液中溶解度更高。如有必要，调节待测溶液（或它的稀释液）的pH值，使鲎试剂和供试品溶液的混合物的pH值在所选鲎试剂生产商的指定pH范围内。一般要求供试品溶液的pH值范围为6.0——8.0。可用鲎试剂生产商推荐的酸、碱或适当的缓冲溶液来调解pH值。酸或碱溶液须用BET检查用水在去除内毒素的容器中配制。缓冲液必须经过验证不含内毒素和干扰因子。
DETERMINATION OF THE MAXIMUM VALID DILUTION确定最大有效稀释倍数
The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample at which the endotoxin limit can be determined. Determine the MVD using the following formulae:
最大有效稀释倍数（MVD）指可检测出内毒素限值的供试品溶液的最大稀释倍数。MVD按下列公式确定：
MVD=
Endotoxin limit × concentration of test solution
λ
MVD = 内毒素限值×供试品溶液浓度/λ
Endotoxin limit: the endotoxin limit for active substances administered parenterally, defined on the basis of dose, is equal to
K
M
内毒素限值：在剂量的基础上，注射药物的活性成分的内毒素限度为K/M
K = threshold pyrogenic dose of endotoxin per kilogram of body mass,
M = maximum recommended bolus does of product per kilogram of body mass.
K=人每公斤体重每小时最大可接受的内毒素剂量（EU/kg）
M＝人用每公斤体重每小时的最大供试品剂量。
When the product is to e injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour period.
The endotoxin limit for active substances administered parenterally is specified in units such as: IU/mL, IU/mg, IU/Unit of biological activity, ect, in monographs.
专论中，注射剂活性成分的内毒素限度的单位有IU/ml、IU/mg、IU/生物活性单位等。
Concentration of test solution:
供试品溶液的浓度表示法：
— mg/mL if the endotoxin limit is specified by mass (IU/mg),
— 当内毒素限度以质量（IU/mg）表示时，用mg/ml表示浓度。
— Units/mL if the endotoxin limit is specified by unit of biological activity (IU/Unit),
— 当内毒素限度以（IU/ml）表示时，用ml/ml表示浓度。
— ml/mL if the endotoxin limit is specified by volume (IU/mL).
— 当内毒素限度以生物单位（IU/Unit）表示时，用Units/ml表示浓度。

λ = the lablelled lysate sensitivity in the gel-clot techniques (IU/mL) or the lowest concentration used in the standard curve of the turbidimetric or chromogenic techniques.
λ=指凝胶法中鲎试剂的标示灵敏度，或指浊度法或显色法使用的标准回归曲线上最低点（IU/ml)的对应值。
7. GEL-CLOT TECHNIQUE (METHODS A AND B) 凝胶法（方法A和B）
The gel-clot techniques allow detection or quantification of endotoxins and is based on clotting of the lysate in the presence of endotoxins. The minimum concentration of endotoxins required to cause the lysate to clot under standard conditions is the labeled lysate sensitivity. To ensure both the precision and validity of the test, confirm the labeled lysate sensitivity and perform the test for interfering factors as described under 1. Preparatory testing.
凝胶法系通过鲎试剂与内毒素产生凝集反应的原理来检测或定量内毒素的方法。在标准条件下，可引起鲎试液凝集的内毒素的最低浓度即为鲎试剂的标示灵敏度。为确保试验结果精确、有效，应根据预备试验中的说明开展试验，复核鲎试剂的标示灵敏度和试验的干扰因素。
1. PREPARATORY TESTING预备试验
(i) Confirmation of the labeled lysate sensitivity鲎试剂的标示灵敏度复核试验
Confirm in 4 replicates the labeled sensitivity λ, expressed in IU/mL, of the lysate solution prior to use in the test. Confirmation of the lysate sensitivity is carried out when a new lot of lysate is used or when there is any change in the test conditions which may affect the outcome of the test.
灵敏度用IU/ml表示。应在鲎试剂用于检查内毒素之前对灵敏度进行复核；复核试验需要制备标示灵敏度λ的4支平行管。当使用新批号的鲎试剂或试验条件发生了任何可能影响检验结果的改变时，应进行鲎试剂灵敏度复核试验。
Prepare standard solutions of at least 4 concentrations equivalent to 2λ, λ, 0.5λ and 0.25λ by diluting the standard endotoxin stock solution with water for BET.
用BET用水稀释内毒素储备标准溶液，制成2λ、λ、0.5λ和0.25λ四种浓度的标准溶液。
Mix a volume of the lysate solution with an equal volume of 1 of the standard solutions (such as 0.1 mL aliquots) in each tube. When single test vials or ampoules containing lyophilized lysate are employed, add solutions of standards directly to the vial or ampoule. Incubate the reaction mixture for a constant period according to the recommendations of the lysate manufacturer (usually at 37±1?C for 60±2 min), avoiding vibration. Test the integrity of the gel: for tubes, take each tube in turn directly from the incubator and invert it through approximately 180? in one smooth motion. If a firm gel has formed that remains in place upon inversion, record the result as positive. A result is negative if an intact gel is not formed.
在每支试管中，分别混合等体积（通常为0.1mL)的鲎试液和标准溶液。如使用的是装有鲎试剂冻干粉末的西林瓶或安瓿，可向其中直接加入溶液。按照鲎试剂生产商的建议，将反应混合物孵育一段时间(通常在37±1℃下保温60±2min)，在此过程中，不能振动试管。凝胶的完整性测试：如使用试管作为容器，先将试管从保温箱中取出，再缓缓将试管倒转180o。如果形成了坚实的凝胶，倒转后凝胶不移位，此时将该项检查的结果记录为阳性。如未能形成坚实且不变形的凝胶，该项检查的结果即为阴性。
The test is considered valid when the lowest concentration of the standard solutions shows a negative result in all replicate tests. The end-point is the lowest concentration in the series of decreasing concentrations of standard endotoxin that clots the lystae. Determine the geometric mean end-point concentration by calculating the mean of the logarithms of the end-point concentrations of the 4 dilution series, take the antilogarithm of this value, as indicated by the following expression:
仅在最低浓度的标准溶液的所有平行管的检查结果均为阴性的情况下，试验方为有效。反应终点浓度指系列递减的内毒素浓度中最后一个呈阳性结果的浓度。将终点浓度取对数，计算它们的平均值，再将平均值的结果取反对数，最后的表达式如下：
Geometric mean end-point concentration = antilog
Σe
f
Σe = sum of the log end-point concentrations of the dilution series used,
f = number of replicates.
终点浓度的几何平均值＝log-1(Σe/f)
Σe＝所用系列溶液的终点浓度的对数值的和
f＝平行管的数量
The geometric mean end-point concentration is the measured sensitivity of the lysate solution (IU/mL). If this is not less than 0.5λ and not more than 2λ, the labeled sensitivity is confirmed and is used in the test performed with this lysate.
反应终点的浓度的几何平均值即为鲎试剂灵敏度的测量值（IU/ml）。当终点浓度的几何平均值在0.5λ至2.0λ之间时，可判定受试鲎试剂的标示灵敏度为λ，可用于内毒素的检查。
(ii) Test for interfering factors干扰因素试验
Prepare solutions A, B, C and D as shown in Table 2.6.14-1, and use the test solutions at a dilution less than the MVD, not containing any detectable endotoxins, operating as described under 1. preparatory testing, (i) Confirmation of the labeled lysate sensitivity.
按表2.6.14.-1制备溶液A、B、C、D。供试品的稀释度不得超过MVD，且供试品溶液不能检查出内毒素，具体操作见（1）预备试验，（i）鲎试剂标示灵敏度的复核试验项。
The geometric mean end-point concentrations of solutions B and C are determined using the expression described in 1. Preparatory testing, (i) Confirmation of the labeled lysate sensitivity.
根据（1）预备试验下的（i）鲎试剂标示灵敏度的复核试验项中列出的公式，计算B和C溶液的终点浓度的几何平均值。
Table 2.6.14.-1
Solution
Endotoxin concentration/solution to which endotoxin is added
Diluent
Dilution factor
Endotoxin concentration
Number of replicates
A
None/Test solution
-
-
-
4
B
2λ/Test solution
Test solution
1
2
4
8
2λ
1λ
0.5λ
0.25λ
4
4
4
4
C
2λ/Water for BET
Water for BET
1
2
4
8
2λ
1λ
0.5λ
0.25λ
2
2
2
2
D
None/Water for BET
-
-
-
2
Solution A = solution of the preparation being examined that is free of detectable endotoxins.
Solution B = test for interference.
Solution C = control of the labeled lysate sensitivity.
Solution D = negative control (water for BET).
表2.6.14-1
溶液
内毒素浓度/配制内毒素的溶液
稀释剂
稀释倍数
稀释后内毒素的浓度
平行管数
A
无/供试品溶液
－
－
－
4
B
2λ/供试品溶液
供试品溶液
1
2
4
8
2λ
1λ
0.5λ
0.25λ
4
4
4
4
C
2λ/BET用水
BET用水
1
2
4
8
2λ
1λ
0.5λ
0.25λ
2
2
2
2
D
0/BET用水
－
－
－
2
A=经检查无内毒素的溶液
B=干扰实验用
C=鲎试剂标示灵敏度的对照品
D=阴性对照品（BET检查用水）
The test for interfering factors must be repeated when any changes are made to the experimental conditions that are likely to influence the result of the test.
如试验条件发生了任何可能影响到试验结果的变化，须重新进行干扰因素试验。
The test is considered valid when all replicates of solutions A and D show no reaction and the result of solution C confirms the labeled lysate sensitivity.
只有当溶液A和D的所有平行管中无反应、且溶液C的结果在复核的鲎试剂灵敏度范围内时，试验方有效。
If the sensitivity of the lysate determined with solution B is not less than 0.5λ and not greater than 2λ, the test solution does not contain interfering factors under the experimental conditions used. Otherwise, the test solution interferes with the test.
经测定，如果溶液B的灵敏度在[0.5λ，2.0λ]间，可判定供试品溶液在该实验条件下，对实验无干扰；反之则判定供试品溶液对试验能形成干扰。
If the preparation being examined interferes with the test at a dilution less than the MVD, repeat the test for interfering factors using a greater dilution, not exceeding the MVD. The use of a more sensitive lysate permits a greater dilution of the preparation being examined and this may contribute to the elimination of interference.
若供试品溶液在小于MVD的稀释倍数下对试验有干扰，应将供试品溶液进行不超过MVD的进一步稀释，再重复干扰试验。使用灵敏度更高的鲎试剂进行内毒素的检查时，因为更高灵敏度鲎试剂能排除实验的干扰因素，供试品的稀释倍数也可适当放宽。
Interference may be overcome by suitable validated treatment, such as filtration, neutralization, dialysis or heat treatment. To establish that the treatment chose effectively eliminates interference without loss of endotoxins, repeat the test for interfering factors using the preparation being examined to which the standard endotoxin has been added and which has then been submitted to the chosen treatment.
可通过其他适宜的方法（如过滤、中和、透析或加热处理等）排除干扰。为确保所选择的处理方法能有效地排除干扰且不会使内毒素失去活性，要使用预先添加了标准内毒素再经过处理的供试品溶液进行干扰实验。
2. LIMIT TEST (METHOD A) 限度试验（方法A）
(i) Procedure方法
Prepare solutions A, B, C and D as shown in Table 2.6.14.-2, and perform the test on these solutions following the procedure described under 1. Preparatory testing, (i) Confirmation of the labeled lysate sensitivity.
按表2.6.14-2制备溶液A、B、C、D。实验方法见（1）预备试验的（i）鲎试剂标示灵敏度的复核试验项。
Table 2.6.14.-2
Solution
Endotoxin concentration/Solution to which endotoxin is added
Number of replicates
A
None/Diluted test solution
2
B
2λ/Diluted test solution
2
C
2λ/Water for BET
2
D
None/Water for BET
2
表2.6.14-2
溶液
内毒素浓度/配制内毒素的溶液
平行管数
A
0/供试品溶液
2
B
2λ/供试品溶液
2
C
2λ/BET用水
2
D
0/BET用水
2

Prepare solution A and solution B (positive product control) using a dilution