CN100383528C - A method for bacterial endotoxin inspection by measuring turbidity value - Google Patents
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Abstract
一种通过测定浊度值来进行细菌内毒素检查的方法，其特征在于：将供试品与鲎试剂等比混合，反应温度为37.0±1℃，反应时间为60±1分钟；检测浊度值，检测浊度值的检测波长范围在400至700nm之间，浊度值定义为：A0’＝-lg(It/I’0)，其中I’0为初始透射光强度，It为检测时透射光强度；浊度值大于等于0.055时，为阳性结果；浊度值小于0.055时，为阴性结果。本发明可以通过专门的仪器来测量实现，减少了人工目测判断凝胶与否的误差，提高了结果的准确性；可望替代常规凝胶法进行细菌内毒素的检查，用于药品质量控制和制剂的生产过程监控等。A method for detecting bacterial endotoxin by measuring turbidity value, characterized in that: the test product is mixed with Limulus reagent in equal proportions, the reaction temperature is 37.0±1°C, and the reaction time is 60±1 minute; the turbidity is detected value, the detection wavelength range of the detection turbidity value is between 400 and 700nm, and the turbidity value is defined as: A 0 '=-lg(I t /I' 0 ), wherein I' 0 is the initial transmitted light intensity, I t It is the transmitted light intensity during detection; when the turbidity value is greater than or equal to 0.055, it is a positive result; when the turbidity value is less than 0.055, it is a negative result. The present invention can be realized by measuring with a special instrument, which reduces the error of manual visual inspection to judge whether it is gel or not, and improves the accuracy of the result; it is expected to replace the conventional gel method for bacterial endotoxin inspection, and is used for drug quality control and Production process monitoring of preparations, etc.
Description
技术领域： Technical field:
本发明涉及细菌内毒素的测定方法，特别提供了一种通过测定浊度值来进行细菌内毒素检查的标准。The invention relates to a method for measuring bacterial endotoxin, and in particular provides a standard for checking bacterial endotoxin by measuring the turbidity value.
细菌内毒素(Bacterial Endotoxin)是革兰氏阴性细菌所产生的具有各种生物活性的大分子物质，其主要化学成分为脂多糖(LPS)。内毒素是注射剂药品(原料等)中的主要污染物质。Bacterial endotoxin (Bacterial Endotoxin) is a macromolecular substance with various biological activities produced by Gram-negative bacteria, and its main chemical component is lipopolysaccharide (LPS). Endotoxin is the main pollutant in injection drugs (raw materials, etc.).
细菌内毒素的检测方法有家兔试验法、鲎试验法。鲎试验法(LimulusAmebocyte Lysate，LAL)是利用鲎试剂与内毒素发生凝集反应的原理，定性或定量检测药品或机体血液中细菌内毒素感染程度的一种体外检测方法。具体作法又分为凝胶法和定量法。The detection methods of bacterial endotoxin include rabbit test method and limulus test method. Limulus Amebocyte Lysate (LAL) is an in vitro detection method that uses the principle of agglutination reaction between Limulus Amebocyte Lysate and endotoxin to qualitatively or quantitatively detect the degree of bacterial endotoxin infection in the blood of the drug or the body. The specific method is divided into gel method and quantitative method.
凝胶法就是一种定性、限量测定内毒素的方法。其原理是在专用的试管中将内毒素样品与鲎试剂等量混合，保温反应，当内毒素与鲎试剂中的C因子发生反应后，激活凝固酶原，切断凝固蛋白原特定位置的精氨酸肽键，形成凝固蛋白从而产生凝胶。中国药典2000年附录中对于细菌内毒素检查法的规定，就是依据此方法，用来判定供试品中内毒素的限量是否符合规定，需要实验者目测是否出现凝胶来判定阴、阳性结果，实验本身工作量较大，且易出现人为误差。The gel method is a method for qualitative and limited determination of endotoxin. The principle is to mix the endotoxin sample with the Limulus reagent in equal amounts in a special test tube and keep warm for reaction. When the endotoxin reacts with factor C in the Limulus reagent, the procoagulase is activated and the arginine at a specific position of the procoagulant is cut off. Acid-peptide bonds form coagulated proteins to produce gels. The provisions of the bacterial endotoxin test method in the appendix of the Chinese Pharmacopoeia in 2000 are based on this method, which is used to determine whether the limit of endotoxin in the test product meets the regulations. The experiment itself has a large workload and is prone to human error.
定量法是利用动态比浊法的原理，即在内毒素与鲎试剂反应过程中，采用专用仪器检测记录其浊度变化，预设一个浊度值，当浊度值变化到该预设值时所需的时间作为特征反应时间，该特征反应时间的对数与内毒素浓度的对数呈线性关系。此方法要求标准曲线的重复性好。The quantitative method is based on the principle of dynamic turbidimetry, that is, during the reaction process of endotoxin and LAL reagent, use a special instrument to detect and record its turbidity change, preset a turbidity value, when the turbidity value changes to the preset value The required time is taken as the characteristic reaction time, and the logarithm of the characteristic reaction time is linearly related to the logarithm of the endotoxin concentration. This method requires good repeatability of the standard curve.
目前凝胶法和定量法两种方法之间还没有比较明确的对应关系，药检机构和生产商在实际工作中主要采用凝胶法，依靠人工进行检查，费时、费力、人为干扰因素较多。At present, there is no clear correspondence between the gel method and the quantitative method. Drug testing agencies and manufacturers mainly use the gel method in actual work, relying on manual inspection, which is time-consuming, laborious, and has many human interference factors.
发明内容： Invention content:
本发明的目的在于提供一种通过测定浊度值来进行细菌内毒素检查的标准方法，该方法操作简便、检测结果准确。The purpose of the present invention is to provide a standard method for bacterial endotoxin inspection by measuring the turbidity value, which is easy to operate and accurate in detection results.
本发明提供了一种通过测定浊度值来进行细菌内毒素检查的方法，其特征在于：The invention provides a method for carrying out bacterial endotoxin inspection by measuring turbidity value, characterized in that:
——将供试品与鲎试剂等比混合(体积)，反应温度为37.0±1℃，反应时间为60±1分钟；—— Mix the test product and Limulus reagent in equal proportions (volume), the reaction temperature is 37.0±1°C, and the reaction time is 60±1 minute;
——检测浊度值，浊度值定义为：A0’＝-lg(It/I0’)，其中I’0为初始透射光强度，It为检测时透射光强度；——Detect the turbidity value, the turbidity value is defined as: A 0 '=-lg(I t /I 0 '), where I' 0 is the initial transmitted light intensity, I t is the transmitted light intensity during detection;
——蚀度值大于等于0.055时，为阳性结果；浊度值小于0.055时，为阴性结果。——When the corrosion value is greater than or equal to 0.055, it is a positive result; when the turbidity value is less than 0.055, it is a negative result.
本发明通过测定浊度值来进行细菌内毒素检查的方法中，对所用的检测仪器要求不是非常严格，所述供试品与鲎试剂的反应在试管中进行，反应液的总体积为0.2毫升至0.7毫升。所采用的试管，其内径可以在5.4毫米至10.2毫米之间。检测浊度值的检测波长范围在400至700nm之间。最好为660nm。In the method for carrying out bacterial endotoxin inspection by measuring the turbidity value of the present invention, the requirements for the detection instrument used are not very strict, and the reaction between the test product and the limulus reagent is carried out in a test tube, and the total volume of the reaction solution is 0.2 ml. to 0.7 ml. The test tube used may have an inner diameter between 5.4 mm and 10.2 mm. The detection wavelength range for detecting the turbidity value is between 400 and 700 nm. Preferably 660nm.
另外本发明通过测定浊度值来进行细菌内毒素检查的方法中，对试剂的要求也不是很高，所采用的鲎试剂，其灵敏度可为0.015-0.5EU/ml。In addition, in the method for detecting bacterial endotoxin by measuring the turbidity value of the present invention, the requirements for reagents are not very high, and the sensitivity of the limulus reagent used can be 0.015-0.5 EU/ml.
本发明的需要检测的关键参数浊度值定义为：A0’＝-lg(It/I0’)，其中I’0为初始透射光强度，It为检测时透射光强度，浊度值与入射光强度无关，因此消除了测定管尺寸、反应体系初始状态的影响。另外，该参数可以容易地通过很多现有的技术手段测量实现，还可以通过专门的仪器来测量实现，减少了人工目测判断凝胶与否的误差，提高了结果的准确性；可望替代常规凝胶法进行细菌内毒素的检查，用于药品质量控制和制剂的生产过程监控等。The key parameter turbidity value that needs detection of the present invention is defined as: A 0 '=-lg(I t /I 0 '), wherein I ' 0 is initial transmitted light intensity, I t is transmitted light intensity when detection, turbidity The value has nothing to do with the incident light intensity, thus eliminating the influence of the measurement tube size and the initial state of the reaction system. In addition, this parameter can be easily measured by many existing technical means, and can also be measured by a special instrument, which reduces the error of manual visual inspection to determine whether the gel is gelled or not, and improves the accuracy of the results; it is expected to replace conventional The gel method is used for the inspection of bacterial endotoxin, which is used for the quality control of pharmaceuticals and the monitoring of the production process of preparations.
附图说明： Description of drawings:
图1为反应体系浊度值变化示意。Figure 1 is a schematic diagram of the change of the turbidity value of the reaction system.
具体实施方式： Detailed ways:
常规的细菌内毒素限量的测定方法是：首先需要进行供试品干扰试验，将细菌内毒素工作标准品稀释到2.0λ、1.0λ、0.5λ和0.25λ浓度，每一稀释液做4管(10mm内径试管)，轻轻振动上述试管混匀内容物，封闭管口，置37±1℃水浴中。保温60±2分钟待观察结果，另取内毒素检查用水和供试品稀释液，各做2支阴性对照管。其最高浓度2.0λ的4管应均为阳性，最低浓度0.25λ的4管应均为阴性，且阴性对照管均为阴性时，则无干扰。可根据供试品内毒素限量选择合适灵敏度的鲎试剂，依照上述方法做试验来检查供试品内毒素是否超出限量。The conventional determination method of bacterial endotoxin limit is: firstly, it is necessary to carry out the interference test of the test product, dilute the bacterial endotoxin working standard to the concentration of 2.0λ, 1.0λ, 0.5λ and 0.25λ, and make 4 tubes for each dilution ( 10mm inner diameter test tube), gently vibrate the above test tube to mix the contents, close the mouth of the tube, and place it in a water bath at 37±1°C. Incubate for 60±2 minutes to observe the results, and then take water for endotoxin inspection and diluent of the test sample, and make 2 negative control tubes for each. The 4 tubes with the highest concentration of 2.0λ should all be positive, the 4 tubes with the lowest concentration of 0.25λ should all be negative, and when the negative control tubes are all negative, there is no interference. According to the endotoxin limit of the test product, the LAL reagent with appropriate sensitivity can be selected, and the test should be carried out according to the above method to check whether the endotoxin of the test product exceeds the limit.
本发明通过测定浊度值来进行细菌内毒素检查的方法是，在内毒素测定仪上，测定含内毒素样品与鲎试剂反应体系的浊度值，当反应时间为60分钟，浊度值大于等于0.055时，为阳性结果；浊度值小于0.055时，为阴性结果。The method for carrying out bacterial endotoxin inspection by measuring the turbidity value of the present invention is to measure the turbidity value of the reaction system between the endotoxin-containing sample and the Limulus reagent on the endotoxin measuring instrument. When the reaction time is 60 minutes, the turbidity value is greater than When it is equal to 0.055, it is a positive result; when the turbidity value is less than 0.055, it is a negative result.
实施例1.Example 1.
常规凝胶法的操作步骤是：将供试品与鲎试剂等比混合于专用试管中，体积为0.2ml，混匀，封闭管口，置37±1℃水浴中，保温60±2分钟，取出试管缓缓倒转180度，观察结果。当试管内凝胶不变形，不从管壁滑脱者为阳性结果；凝胶不能保持完整，并从管壁滑脱者为阴性结果。The operation steps of conventional gel method are: mix the test product and Limulus reagent in equal proportions in a special test tube with a volume of 0.2ml, mix well, seal the tube mouth, put it in a water bath at 37±1°C, and keep it warm for 60±2 minutes. Take out the test tube and slowly turn it upside down 180 degrees to observe the result. When the gel in the test tube is not deformed and does not slip off the tube wall, it is a positive result; if the gel cannot remain intact and slips off the tube wall, it is a negative result.
在内毒素测定仪上，将供试品与鲎试剂等比混合于7mm内径平底试管，反应温度为37.1℃，反应总体积为0.3ml，鲎试剂灵敏度为0.06EU/ml，供试品为标准内毒素溶液，浓度为0、0.03、0.06、0.12EU/ml，检测时间达60分钟时，其对应的浊度值分别为0.005、0.024、0.055和0.090；该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.005和0.024时，凝胶法的结果为阴性；浊度值为0.055和0.090时，凝胶法的结果为阳性。On the endotoxin analyzer, mix the test product and Limulus reagent in equal proportions in a 7mm inner diameter flat-bottomed test tube, the reaction temperature is 37.1°C, the total reaction volume is 0.3ml, the sensitivity of the Limulus reagent is 0.06EU/ml, and the test product is used as the standard Endotoxin solution, the concentration is 0, 0.03, 0.06, 0.12EU/ml, when the detection time reaches 60 minutes, the corresponding turbidity values are respectively 0.005, 0.024, 0.055 and 0.090; The gel method was tested in parallel, and the results showed that: when the turbidity value was 0.005 and 0.024, the result of the gel method was negative; when the turbidity value was 0.055 and 0.090, the result of the gel method was positive.
实施例2.Example 2.
在内毒素测定仪上将供试品与鲎试剂等比混合于5.4mm内径平底试管，反应温度为37.2℃，反应总体积为0.3ml，鲎试剂灵敏度为0.5EU/ml，供试品为含有内毒素的去离子水，反应时间达15分钟时，检测其对应的浊度值为0.063；该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.063，凝胶法的结果为阳性。On the endotoxin analyzer, mix the test product and Limulus reagent in equal proportions in a 5.4mm inner diameter flat-bottomed test tube, the reaction temperature is 37.2°C, the total reaction volume is 0.3ml, the sensitivity of the Limulus reagent is 0.5EU/ml, and the test product contains For deionized water of endotoxin, when the reaction time reaches 15 minutes, the corresponding turbidity value is detected to be 0.063; the test product and Limulus reagent are tested in parallel according to the gel method, and the results show that the turbidity value is 0.063, The result of the gel method was positive.
实施例3.Example 3.
在内毒素测定仪上将供试品与鲎试剂等比混合于采用5.4mm内径平底试管，反应温度为37.1℃，反应总体积为0.3ml，鲎试剂灵敏度为0.03EU/ml，供试品为浓度为0.24EU/ml标准内毒素水溶液，检测时间达60分钟时，其对应的浊度值为0.07；该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.07，凝胶法的结果为阳性。On the endotoxin analyzer, the test product and the LAL reagent are mixed in equal proportions in a flat-bottomed test tube with an inner diameter of 5.4mm, the reaction temperature is 37.1°C, the total reaction volume is 0.3ml, the sensitivity of the LAL reagent is 0.03EU/ml, and the test product is The concentration is 0.24EU/ml standard endotoxin aqueous solution, and when the detection time reaches 60 minutes, the corresponding turbidity value is 0.07; the test sample and Limulus reagent are tested in parallel according to the gel method, and the results show that: the turbidity value 0.07, the result of the gel method was positive.
实施例4.Example 4.
在内毒素测定仪上将供试品与鲎试剂等比混合于采用7mm内径平底试管，反应温度为37.1℃，反应总体积为0.5ml，鲎试剂灵敏度为0.03EU/ml，供试品为含有内毒素的去离子水，当检测时间达25分钟时，其浊度值为0.063时，采用分光光度计对其在400、600和700nm三个检测波长下进行测定，结果吸光度值分别为0.444、0.166和0.151。该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.063时，凝胶法的结果为阳性。On the endotoxin analyzer, mix the test product and Limulus reagent in equal proportions in a flat-bottomed test tube with an inner diameter of 7mm, the reaction temperature is 37.1°C, the total reaction volume is 0.5ml, the sensitivity of the Limulus reagent is 0.03EU/ml, and the test product contains For the deionized water of endotoxin, when the detection time reaches 25 minutes, when its turbidity value is 0.063, use a spectrophotometer to measure it under three detection wavelengths of 400, 600 and 700nm, and the resultant absorbance values are respectively 0.444, 0.166 and 0.151. The test product and Limulus reagent were tested in parallel according to the gel method, and the results showed that when the turbidity value was 0.063, the result of the gel method was positive.
实施例5.Example 5.
采用分光光度计，在22.℃的室温下，采用10.2mm内径的试管作为反应测定管，供试品为含有内毒素的去离子水样品，鲎试剂灵敏度为0.5EU/ml，测定体系体积为0.7ml，供试品与鲎试剂的比例为1∶1，检测波长设定为600nm，测定其吸光度值，85分钟时，吸光度值为0.124。Using a spectrophotometer, at a room temperature of 22°C, a test tube with an inner diameter of 10.2mm is used as a reaction measurement tube, the test product is a deionized water sample containing endotoxin, the sensitivity of the limulus reagent is 0.5EU/ml, and the volume of the measurement system is 0.7ml, the ratio of the test product and the limulus reagent is 1: 1, the detection wavelength is set at 600nm, and its absorbance value is measured. In 85 minutes, the absorbance value is 0.124.
在内毒素测定仪上将供试品与鲎试剂等比混合于采用7mm内径平底试管，反应温度为37.1℃，反应总体积为0.7ml，鲎试剂灵敏度为0.5EU/ml，检测时间达85分钟时，其对应的浊度值为0.10。该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.10时，凝胶法的结果为阳性。On the endotoxin tester, mix the test product and Limulus reagent in equal proportions in a flat-bottomed test tube with an inner diameter of 7mm. The reaction temperature is 37.1°C, the total reaction volume is 0.7ml, the sensitivity of Limulus reagent is 0.5EU/ml, and the detection time is 85 minutes. When , its corresponding turbidity value is 0.10. The test product and Limulus reagent were tested in parallel according to the gel method, and the results showed that when the turbidity value was 0.10, the result of the gel method was positive.
实施例6.Example 6.
在内毒素测定仪上，将1.5mg/ml的克林霉素磷酸酯氯化钠注射液与鲎试剂等比混合于7mm内径平底试管，反应温度为37.0℃，鲎试剂灵敏度为0.125EU/ml，反应体积0.3ml，60分钟时，测定浊度值为0.004。同时该供试品与鲎试剂依照凝胶法做平行对照检测，结果表明：浊度值为0.004时，凝胶法的结果为阴性。On the endotoxin analyzer, mix 1.5mg/ml clindamycin phosphate sodium chloride injection and Limulus reagent in equal proportions in a 7mm inner diameter flat bottom test tube, the reaction temperature is 37.0°C, and the sensitivity of Limulus reagent is 0.125EU/ml , the reaction volume was 0.3ml, and at 60 minutes, the measured turbidity value was 0.004. At the same time, the test product and Limulus reagent were tested in parallel according to the gel method, and the results showed that: when the turbidity value was 0.004, the result of the gel method was negative.
实施例7.Example 7.
在内毒素测定仪上将供试品与鲎试剂等比混合于5.4mm内径平底试管，反应温度为37.0℃，反应总体积为0.2ml，鲎试剂灵敏度为0.25EU/ml，供试品为含有内毒素的去离子水，检测时间达60分钟时，其对应的浊度值为0.14；该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.14时，凝胶法的结果为阳性。On the endotoxin analyzer, mix the test product and Limulus reagent in equal proportions in a 5.4mm inner diameter flat bottom test tube, the reaction temperature is 37.0°C, the total reaction volume is 0.2ml, the sensitivity of the Limulus reagent is 0.25EU/ml, and the test product contains The deionized water of endotoxin, when the detection time reaches 60 minutes, its corresponding turbidity value is 0.14; this test product and Limulus reagent are carried out parallel control detection according to the gel method at the same time, the result shows: when the turbidity value is 0.14, The result of the gel method was positive.
实施例7Example 7
在内毒素测定仪上将供试品与鲎试剂等比混合于7mm内径平底试管，反应温度为37.0℃，反应总体积为0.3ml，鲎试剂灵敏度为0.06EU/ml，供试品为氯化钠水溶液，浓度为140mM，检测时间达60分钟时，其对应的浊度值为0.004，该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.004时，凝胶法的结果为阴性。On the endotoxin analyzer, mix the test product and Limulus reagent in equal proportions in a 7mm inner diameter flat-bottomed test tube, the reaction temperature is 37.0°C, the total reaction volume is 0.3ml, the sensitivity of the Limulus reagent is 0.06EU/ml, and the test product is chlorinated Sodium aqueous solution, concentration is 140mM, when detection time reaches 60 minutes, its corresponding turbidity value is 0.004, this test sample and Limulus reagent are carried out parallel control detection according to gel method at the same time, the result shows: when turbidity value is 0.004, The gel method was negative.
实施例8Example 8
在内毒素测定仪上将供试品与鲎试剂等比混合于7mm内径平底试管，反应温度为37.5℃，反应总体积为0.3ml，鲎试剂灵敏度为0.015EU/ml，供试品为无热源水，检测时间达60分钟时，其对应的浊度值为0.003；该供试品与鲎试剂同时依照凝胶法做平行对照检测，结果表明：浊度值为0.003时，凝胶法的结果为阴性。On the endotoxin analyzer, mix the test product and Limulus reagent in equal proportions in a 7mm inner diameter flat-bottomed test tube, the reaction temperature is 37.5°C, the total reaction volume is 0.3ml, the sensitivity of the Limulus reagent is 0.015EU/ml, and the test product is no pyrogen Water, when the detection time reaches 60 minutes, its corresponding turbidity value is 0.003; the test sample and Limulus reagent are tested in parallel according to the gel method at the same time, and the results show that: when the turbidity value is 0.003, the result of the gel method is negative.
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| CN101477103B (en) * | 2009-01-05 | 2012-06-27 | 范能全 | Full-pyrogen detection method for pyrogenicsubstance content in injection |
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| CN1132352A (en) * | 1994-10-31 | 1996-10-02 | 和光纯药工业株式会社 | Process for measuring amount of endotoxin or (1-3)-beta-D-glucan by kinetic turbidimetric method |
| WO2004001419A1 (en) * | 2002-06-21 | 2003-12-31 | Hemofarm Koncern A.D. Pharmaceutical And Chemical Industry | Process for determining endotoxin level in hemoglobin solutions |
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| US4740460A (en) * | 1984-06-27 | 1988-04-26 | Wako Pure Chemical Industries, Ltd. | Process for measuring endotoxin and apparatus used therefor |
| EP0347951A2 (en) * | 1984-06-27 | 1989-12-27 | Wako Pure Chemical Industries, Ltd. | Apparatus for measuring endotoxin |
| CN1132352A (en) * | 1994-10-31 | 1996-10-02 | 和光纯药工业株式会社 | Process for measuring amount of endotoxin or (1-3)-beta-D-glucan by kinetic turbidimetric method |
| WO2004001419A1 (en) * | 2002-06-21 | 2003-12-31 | Hemofarm Koncern A.D. Pharmaceutical And Chemical Industry | Process for determining endotoxin level in hemoglobin solutions |
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