Comparative evaluation of the human whole blood and human peripheral blood monocyte tests for pyrogens

Two different in vitro tests for pyrogens, using human peripheral blood monocytes (PBMNC) and diluted whole blood (WBC), respectively, were applied to different classes of parenteral medicinal products. Many of these products did not have a specified endotoxin limit concentration that was established as the maximum valid dilution to comply with the test. The results of the in vitro tests for pyrogens were compared with the results from the Limulus amoebocyte lysate (LAL) and rabbit pyrogen tests. The Second International Standard for endotoxin was used to calibrate all of the assays and the International Standard for IL-6 was used to calibrate the IL-6 ELISA which provided the readout for the in vitro tests for pyrogens. Preparatory tests were conducted to ensure that the "criteria for validity and precision of the standard curve" were satisfied and that the drugs being tested did not interfere in the tests. The PBMNC/IL-6 test had a detection limit of 0.06 EU/ml and spike recoveries were 62-165%. The whole blood/IL-6 test also had a detection limit of 0.06 EU/ml and spike recoveries were 58-132%. The application to the detection of non-endotoxin pyrogens needs to be evaluated in more detail, but the two in vitro tests for pyrogens showed good agreement overall, both with each other and with the LAL test and the rabbit pyrogen test for the detection of endotoxins.     

A highly sensitive pyrogen test for antibiotics I: detection of trace amounts of endotoxin in injectable sodium ampicillin preparations

The rabbit pyrogen test (specified in the pharmacopeia) and the Limulus amebocyte lysate (LAL) test are influenced by high concentrations of certain antibiotics. Therefore, it has not been possible to detect trace amounts of endotoxin which may contaminate these antibiotics. To detect trace amounts of endotoxin in injectable sodium ampicillin, an ultrafiltration technique was utilized which removed the antibiotic and left a solution which contained predominantly the endotoxin. After ultrafiltration, a trace amount of pyrogen (which otherwise could not be detected) was found using both the rabbit pyrogen and the LAL tests. The endotoxin was also determined quantitatively using a chromogenic endotoxin reagent which is made by combining the Limulus amebocyte lysate and a synthetic substrate with a suitable chromophore.     

注射用盐酸普鲁卡因细菌内毒素检查的方法研究

目的考察注射用盐酸普鲁卡因的细菌内毒素检查的方法.方法按中国药典2000年版二部收载的细菌内毒素检查方法进行试验,并与热原检查法进行对比试验.结果注射用盐酸普鲁卡因对鲎试剂的凝聚反应无干扰作用.结论可以用细菌内毒素检查法替代家兔热原法来控制注射用盐酸普鲁卡因的质量.     

注射用阿莫西林钠克拉维酸钾细菌内毒素检查的方法研究

目的：注射用阿莫西林钠克拉维酸钾为一抗生素复方制剂,厂家内控质量标准中控制细菌内毒素的方法为动力学浊度法,内毒素限值为0.29EU/mg。USP23,USP24及BP1998均未收载本品的注射剂,而中国药典1995年版1998增补已收载,但控制细菌内毒素的方法为家兔热原检查法,剂量为20mg/kg。为了在国内开展本品的细菌内毒素检查,我们对本品进行了细菌内毒素检查凝胶法的方法研究。方法：按中国药典1995年版1998增补本细菌内毒素检查进行干扰实验和结果判断。结果：本品在0.12至2.0mg/ml的浓度范围内。对细菌内毒素与鲎试剂的反应无干扰作用,确定本品细菌内毒素限值为0.25EU/mg。结论：按0.25EU/mg限值我们对两个国家进口的5批样品进行了检验,并与家兔热原检查法进行了对比,结果一致．都为阴性,认为本品可用细菌内毒素检查凝胶法代替家兔热原检查法。     

The effects and properties of sodium nucleinate as a pyrogen working-standard. 9. Pyrogen detection with epinephrine-skin-, dactinomycin- and LAL-tests. The suitability of sodium nucleinate as a pyrogen standard]

Sodium nucleinate (NN) as well as bacterial lipopolysaccharide (LPS) can be detected by epinephrine-skin, dactinomycin and LAL tests. In the quantitative determination of two pyrogen standards for rabbit tests, consisting of NN, a smaller value was found by LAL test for the standard of greatest pyrogenic effect than for that less pyrogenically effective in rabbits. A standard consisting of NN can be used for the pyrogen test in rabbits. But in the future, if necessary a standard consisting of endotoxin will be used, due to its better comparability of results obtained by LAL and rabbit tests.     

The application of the endotoxin test for globulin and other blood products]

The application of the endotoxin test for globulin and other blood products were investigated by two different limulus amebocyte lysate (LAL) test methods, colorimetric and kinetic turbidimetric methods, using two endotoxin-specific reagents. By the dilution of the blood products in 40 times or more, spiked endotoxin in the products was recovered accurately showing neither inhibition nor enhancement. The definite difference was not shown between the results obtained by the two LAL test methods. According to the method of the endotoxin test described under General Tests of The Japanese Pharmacopeia (thirteenth edition), JPXIII, the maximum valid dilution (MVD) for these products will be calculated to be 40 or more, so it is capable to measure the endotoxin limit for each product. This study indicates that the endotoxin test is applicable to measure the endotoxin content in globulin and other blood products as an alternative method for the rabbit pyrogen test.     

应用LAL动态浊度法测定复方苦参注射液中细菌内毒素含量

目的：建立应用美洲鲎试剂LAL的动态浊度法定量检测复方苦参注射液中细菌内毒素试验方法。方法：采用《中国药典》2015年版(四部)中的细菌内毒素检查法。结果：复方苦参注射液用细菌内毒素定量法检测无干扰因素影响,内毒素回收率在50%～200%范围内。结论：使用LAL进行动态浊度法定量检测复方苦参注射液的细菌内毒素是可行的,可用细菌内毒素方法代替兔热原检查法,同时又避免了限量法内毒素检测时的干扰。     

热原检查中家兔注射细菌内毒素后的升温情况考察

目的:考察细菌内毒素剂量与热原检查家兔法家兔体温的关系。方法:选择未进行任何实验的家兔,每1 kg家兔体质量分别静脉注射细菌内毒素0.25、0.5、1.0、2.0、2.5、5.0、10.0 EU,观察家兔注射细菌内毒素后的升温情况。结果:细菌内毒素注射剂量在0.5 EU/kg之下时,家兔的体温稳定,在0.5 EU/kg以上的细菌内毒素注射剂量时,家兔呈现不同温差的升温度数,注射剂量超过2 EU/kg时,每组有至少一只家兔升温超过0.6℃。结论:热原检查家兔法内毒素剂量>5 EU/kg,家兔总体均有明显升温;内毒素注射剂量与家兔升温度数存在剂量关系。     

The effect of the lipid A analog, E5531 on fever induced by endotoxin from Escherichia coli.

E5531 is a specific lipid A antagonist and is expected to be a drug for the treatment of septic shock. The LAL (limulus amebocyte lysate) activity and pyrogenicity of E5531 were determined. The LAL activity of E5531 is large and confirmed that E5531 had a high affinity to the lipopolysaccharide receptor. The pyrogenic activity of E5531 is weak and the pyrogenic profile of E5531 is different from that of USP (United States Pharmacopoeia) reference standard endotoxin (ETX). E5531 suppressed the pyrogenicity of ETX in rabbits, indicating that E5531 is expected to be useful for the treatment of fever induced by ETX.     

先锋必(注射用头孢哌酮钠)热原检查法和细菌内毒素检查法的比较性研究

本文阐述了应用热原检查法和细菌内毒素检査法同时对120批先锋必(注射用头孢哌酮钠)样品进行检查,并设立多项对比试验,以确定细菌内毒素检查法取代先锋必热原检查的可行性。结果发现,120批先锋必样品经热原检查和细菌内毒素检查均合格,样品内毒素含量远低于美国药典限值。通过测定加有一定量细菌内毒素的先锋必样品组及对照组的热原反应和鲎试验反应发现,在细菌内毒素浓度为10EU/Mし时,対照组可引发很强的热原反应,而先锋必样品(100MG/ML/KG)组所引发的热原反应明显减弱。说明先锋必在高浓度对热原反应有明显抑制作用。同样,本试验也发现,先锋必在较髙浓度对鲎试验也有明显抑制作用,其最高非抑制浓度为2.5MG/ML。对先锋必内毒素限值测定結果发现,按中国药典兔剂量注射,先锋必内毒素含量达0.1EU/MG时即可引发热原反应,而在0.2EU/MG时热原反应明显。本研究证实,细菌内毒素检査法可以代替热原检查法用于先锋必(注射用头孢哌酮钠)检查,中国药典90版规定的注射用头孢哌酮钠热原检查的兔剂M偏高,对热原反应有抑制作用。     

长春西汀葡萄糖注射液细菌内毒素检查法的研究

目的建立长春西汀葡萄糖注射液的细菌内毒素检查法。方法参照2005年版《中国药典（二部）》细菌内毒素检查法进行试验。结果样品原液对鲎试剂的凝聚反应有抑制作用,经2倍稀释可消除干扰因素,将细菌内毒素限值定为0.5 EU/mL,可用灵敏度为0.25 EU/mL的鲎试剂进行检查。结论可用细菌内毒素检查法（凝胶法）替代家兔热原检查法控制产品热原。     

Uridine-induced hyperthermia in the rabbit

Uridine injection in 0.6% saline elevated rabbit temperatures (mean = 0.9 °C) in the USP XX pyrogen test. Hyperthermia was delayed in onset and peaking 3–4 h post injection, but the injection was negative in the limulus amoebocyte lysate (LAL) assay. Uridine from five lots of different sources exceeded USP XX limits in the rabbit pyrogen test and proved negative in the LAL assay. Because the dose of uridine was high, several procedures were used to determine if an impurity was the cause of temperature elevation. Uridine remained pyrogenic in spite of ultrafiltration (10 000 nominal mol. wt), recrystallization and preparative scale HPLC. Sterile filtration and autoclaving also did not affect the response. Hyperthermia, therefore, appears to be an inherent property of uridine. Uridine was also found to release endogenous pyrogen in-vitro from human mononuclear cells. Uridine has been reported to induce fever in man, thus the USP rabbit pyrogen test predicted for the clinical response.     

A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing

Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured. The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC). The detection limit of MM6 for lipopolysaccharide (LPS) and Staphylococcus aureus was comparable to that of MNC. Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6. The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay. As expected, S. aureus and C. albicans did not show any LAL activity. A. niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells. In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S. aureus and S. typhimurium, but the MM6 assay could not detect A. niger, C. albicans and Influenza virus.     

高丽参注射液的细菌内毒素检查法

目的: 建立高丽参注射液的细菌内毒素检测法.方法: 根据中国药典2000版二部收载的细菌内毒素检查的要求进行实验.结果: 高丽参注射液经稀释后可消除对鲎试剂的干扰,用灵敏度为0.5 EU·ml-1的鲎试剂检测内毒素是有效的,并与家兔法的结果一致.结论: 对高丽参注射液可用内毒素检查法替代家兔法的热原检查.     

检测热原的新方案

综述了利用外致热原激活体内血单核细胞（ＭＮＣ）释放细胞因子如白介素－１（ＩＬ－１）、肿瘤坏死因子（ＴＮＦ－α）等即所谓的内致热原的原理，将待检样品与ＭＮＣ共同培养，检测其上清液中的ＩＬ－１或ＴＮＦ－α含量，这一体外检测致热原的新方法。     

动态浊度法检测三种自制注射液中的细菌内毒素

目的:通过考察含糖平衡盐、替硝唑葡萄糖和氧氟沙星葡萄糖注射液对鲎试剂的干扰作用,探讨以细菌内毒素检查法中的凝胶法取代热原检查法检查含糖平衡盐等3种自制注射液细菌内毒素限值的可行性.方法:采用动态浊度法,用EDS-99细菌内毒素测定仪对含糖平衡盐、替硝唑葡萄糖和氧氟沙星葡萄糖注射液进行干扰试验并用凝胶法进行验证.结果:用两个厂家生产的鲎试剂以动态浊度法检测3个不同批号的含糖平衡盐和替硝唑葡萄糖注射液,样品不经稀释,其细菌内毒素回收率均在50%～200%范围内,标准曲线的相关系数绝对值大于0.980,表明含糖平衡盐、替硝唑葡萄糖注射液对鲎试剂反应无干扰作用,3批样品同时用凝胶法检查,其结果均为阴性;氧氟沙星葡萄糖注射液对鲎试剂有干扰,稀释4倍可消除干扰.结论:含糖平衡盐、替硝唑葡萄糖注射液对鲎试剂反应无干扰作用,凝胶法可作为生产含糖平衡盐和替硝唑葡萄糖注射液时日常检查其细菌内毒素限值的方法;氧氟沙星葡萄糖注射液稀释4倍后可用凝胶法检查.     

银杏达莫注射液细菌内毒素检查法的研究

目的建立银杏达莫注射液的细菌内毒素检查法。方法按《中国药典》2005年版(二部)收载的细菌内毒素检查方法及指导原则进行实验。将银杏达莫注射液经64倍稀释,用标示灵敏度为0.5Eu.ml-1的鲎试剂检测其细菌内毒素。结果银杏达莫注射液稀释64倍对鲎试剂无干扰作用。结论可以用细菌内毒素检查法对银杏达莫注射液进行热原检查。     

重组人红细胞生成素注射液中细菌内毒素凝胶检查法的研究

目的本文介绍了一种重组人红细胞生成素注射液的细菌内毒素检查法。方法应用鲎法检查重组人红细胞生成素注射液中的细菌内毒素,考察重组人红细胞生成素注射液对细菌内毒素检查法的干扰。结果重组人红细胞生成素注射液稀释150倍对细菌内毒素检查法无干扰。结论使用凝胶法检查重组人红细胞生成素注射液中的细菌内毒素是可行的,可用细菌内毒素凝胶法代替家兔热原检查法。     

动态浊度法定量检测大蒜注射液中细菌内毒素

目的：分析研究大蒜注射液对鲎试剂的干扰作用，探讨以细菌内毒素检查法取代热原检查法检测大蒜注射液的可行性。方法：本试验采用检测细菌内毒素的动态浊度法，用EDS－99细菌内毒素测定仪对大蒜注射液进行了抑制增强试验。结果：样品经1：20倍衡释，平均回收率在50％－200％范围内，曲线的回归方程为logT=2.8247=0.32525logC,r=-0.99875，表明样品溶液对TAL试剂反应无干扰作用。结论：凝胶法检测大蒜注射液，干扰作用较大。采用动态浊度法进行定量检测，干扰因素小，可以作为日常检测使用。     

20%甘露醇注射液细菌内毒素检查方法的研究

甘露醇注射液是临床上常用的药物,其热原检查方法药典规定为家兔法,我们通过干扰评价实验,证明甘露醇注射液对细菌内毒素检查法干扰,但通过衡释可以消除这种干扰,同时考察10批甘露醇注射液,与热原检查相比较,结果完全一致。