四种氨基酸原料细菌内毒素检查法的研究

依据中国药典2000年版“细菌内毒素检查法”,通过鲎试剂的干扰试验,研究注射用氨基酸原料中细菌内毒素检查法的可行性。结果在2~16倍稀释级下,L-缬氨酸(c=2.5%)和L-异亮氨酸(c=2.5%)对鲎试剂无干扰,检测细菌内毒素的鲎试剂灵敏度≤5.0×102EU.L-1,而在16倍稀释级范围内,L-脯氨酸(c=4.5%)和L-亮氨酸(c=2.0%)对鲎试剂检查法有抑制作用。结论为注射用L-异亮氨酸和L-缬氨酸用灵敏度为5.0×102EU.L-1的鲎试剂检查其细菌内毒素方法可行,可以代替家兔法检查热原。     

动态浊度法定量检测静脉注射用人免疫球蛋白中细菌内毒素含量

用鲎试剂动态浊度法定量检测静脉注射用人免疫球蛋白中细菌内毒素含量并与家兔法检测热原的结果进行对比。根据《中华人民共和国》2005年版三部附录中的细菌内毒素动态浊度法制定内毒素限值,用ATi320-06型动态试管仪和该仪器配置的中文软件"生物探针-2002"分析检测结果。静脉注射用人免疫球蛋白经稀释消除了干扰因素,动态浊度法能定量检测出内毒素含量。实验满足2005年版中国药典要求:标准曲线相关系数的绝对值︱r︱≥0.980,回收率:50%≤R≤200%,变异系数符合鲎试剂厂家的建议值CV%10%。检测结果准确、灵敏、稳定、重现性好,与家兔法检测结果相一致。鲎试剂动态浊度法能定量检测静脉注射用人免疫球蛋白中细菌内毒素含量。     

DNA疫苗的细菌内毒素检测

目的 :考察DNA疫苗细菌内毒素的检查方法的可行性及DNA疫苗的干扰作用。方法 :干扰试验和对比试验。结果 :供试品阴性对照系列样品溶液无干扰作用 ,与家兔法结果一致。结论 :将疫苗稀释 2倍可用灵敏度为 0 5EU ml的鲎试剂作细菌内毒素检查。     

静注人免疫球蛋白细菌内毒素检查方法的建立

目的 :建立用于静脉注射用人免疫球蛋白 (IVIG)的细菌内毒素检查方法。方法 :通过干扰评价实验证明IVIG对细菌内毒素检查法有强烈的抑制作用 ,单纯用简单稀释和调整pH值法无法消除干扰 ,如果用稀释剂 (Ⅰ )对IVIG作 1∶4以上的稀释 ,即可消除样品对该检查法的干扰 ,利用灵敏度为 0 1 2 5EU ml的鲎试剂 ,将样品用稀释剂 (Ⅰ )稀释 4倍 ,按中国药典 ( 2 0 0 0版 )进行细菌内毒素的检查 ,结果与家兔热原质试验进行比较。结论 :IVIG用稀释剂 (Ⅰ )进行适当稀释后 ,可以用细菌内毒素检查法替代家兔热原试验 ,应用于生产过程及半成品的质量控制。     

静脉注射用人免疫球蛋白细菌内毒素检测

探索静脉注射用人免疫球蛋白(IVIG)细菌内毒素含量的鲎试验检测方法。根据《中国药典》(2000版)细菌内毒素含量的检测方法进行,将待检品用NaOH调pH至中性,稀释至2.4倍可用标示灵敏度为0.25EU/m l的特异性鲎试剂进行细菌内毒素检测,结果与家兔法进行比较,并且在样品中加入定量内毒素0.6 EU/m l用两种方法进行对比试验。结果表明用细菌内毒素检测法(鲎试验法)检测静脉注射用人免疫球蛋白是可行的。     

头孢西丁钠细菌内毒素考察

采用两个厂家鲎试剂,对三批供试品(20121201、10121202、20121203)进行了干扰实验。结果表明,本品在有效浓度为1.0mg/ml时,即对细菌内毒素检查无干扰作用。     

动态浊度法鲎试剂在23价肺炎球菌多糖疫苗中间产品内毒素检测中的应用探讨

本文通过使用ATi细菌内毒素动态检测仪进行鲎试剂灵敏度复核试验、干扰试验(回收率试验)和23价肺炎球菌多糖疫苗中间产品内毒素检查,对所得试验结果进行数据分析,找出了影响鲎试剂准确性、灵敏性的干扰因素,从而对鲎试剂的正确使用进行探讨,确保该检测方法的专属性、灵敏度,保证检测结果准确可靠。     

刺五加注射液的细菌内毒素检查探讨

细菌内毒素检查法与热原检查法相比 ,成本低、快速简便、灵敏度高。为探讨采用细菌内毒素检查法检测刺五加注射液热原的可行性 ,我们进行了鲎试剂灵敏度复核试验、干扰试验 ,结果表明刺五加注射液稀释 2 .85倍 ,用灵敏度为 0 .2 5 EU·ml-1的鲎试剂作细菌内毒素检查 ,仍有干扰。1　实验材料　　鲎试剂 (批号 0 30 4 1 9,标示灵敏度 0 .5 EU·ml-1;批号 0 30 5 0 3,标示灵敏度 0 .2 5 EU· ml-1,福州新北生化工业有限公司 ) ;细菌内毒素工作标准品 (批号 2 0 0 30 3,规格 1 5 0 EU·支 -1,中国药品生物制品检定所 ) ;鲎试剂溶解水 (批号 …     

黑皮蛇、山白菜抗细菌内毒素的实验研究

目的探讨黑皮蛇、山白菜的抗细菌内毒素作用。方法采用鲎试剂凝胶法对黑皮蛇、山白菜抗细菌内毒素作用进行实验研究。结果在灵敏度为0.5EU/ml鲎试剂试验中,黑皮蛇、山白菜药液浓度1g/ml,均有抗细菌内毒素作用;不同药液浓度试验,抗细菌内毒素最低药液浓度:黑皮蛇为0.1g/ml,山白菜为0.05g/ml。结论黑皮蛇、山白菜均有较强的抗细菌内毒素作用。     

苦碟子注射液细菌内毒素检查法的探讨

目的：建立苦碟子注射液静脉点滴用药时细菌内毒素的检查方法。方法：采用2个厂家的鲎试剂对苦碟子注射液进行干扰试验。结果：鲎试剂浓度为0．25EU／mL时,苦碟子注射液稀释至1／30对细菌内毒素测定无干扰。结论：鲎试剂法检测苦碟子注射液静脉点滴用药时的细菌内毒素是可行的。     

Comparison of limulus amebocyte lysates and correlation with the United States Pharmacopeial pyrogen test.

Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas.     

Comparison of limulus amebocyte lysates and correlation with the United States Pharmacopeial pyrogen test.

Six limulus amebocyte lysate (LAL) preparations obtained from five different suppliers were evaluated for sensitivity, dependability, cost, convenience of use, and correlation with the United States Pharmacopeial (USP) rabbit pyrogen test method. Endotoxins from various gram-negative microorganisms were used for the evaluation. Major differences among the LAL preparations lie in the area of sensitivity. Differences, up to 100-fold, exist in the sensitivity of the various LAL preparations to the same endotoxin. The LAL tests in general were 3 to 300 times more sensitive than was the USP rabbit pyrogen test method. The LAL and the USP rabbit pyrogen test data correlated well when the endotoxin in a relatively pure and undegraded form was examined. However, large discrepancies in correlation were found when partially degraded endotoxins were compared. One LAL preparation responded to both intact and degraded endotoxin, whereas others responded only to intact endotoxin; the latter closely correlated with the febrile response of the rabbit. Therefore, proper selection of an LAL preparation is important for its application in clinical, pharmaceutical, public health, and environmental areas.     

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.     

Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide.

The rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay have been used to detect endotoxins in vaccines, but interactions between the endotoxins and proteins or aluminum hydroxide can interfere with the results. Currently, the rabbit pyrogen test is used to detect endotoxin in hepatitis B (HB) vaccines even though the HB surface protein, the active ingredient, is over-expressed in and purified from eukaryotic cells which lack endotoxin. Therefore, we examined the possibility of replacing the animal tests with the more efficient LAL test. To this end, we determined whether the aluminum hydroxide in the HB vaccines affects the rabbit pyrogen test and the LAL assay. HB vaccines and HB protein solutions spiked with lipopolysaccharide (LPS) produced almost the same dose-dependent temperature rise in rabbits, indicating that the aluminum hydroxide in the HB vaccine does not interfere with the pyrogenic response in rabbit. In contrast, a spike recovery study showed that aluminum hydroxide interfered with the LAL clot and kinetic assays; however, the LAL clot assay was effective at detecting endotoxin without loss of LAL activity after serial dilution of the samples. Furthermore, there was good correlation in the LAL clot assay between the amount of LPS added and the amount recovered. However, both turbidimetric and chromogenic kinetic assays displayed no correlation between the LPS amount added and recovered. Our results suggest that the LAL clot assay is sensitive and reliable when samples are properly prepared, and can be used to replace the rabbit pyrogen test for the detection of endotoxin in HB vaccines.     

Saline and buffers minimize the action of interfering factors in the bacterial endotoxins test

The bacterial endotoxins test (BET) is the most sensitive assay for measuring endotoxin levels in solution. However, it is difficult to quantify endotoxin levels in some solutions because unknown interfering factors may inhibit or enhance the Limulus amebocyte lysate (LAL) coagulation reaction. We investigated the mechanisms of this interference and found that interference can be reduced or totally suppressed by preparing sample solutions in saline, Dulbecco’s phosphate-buffered saline (D-PBS), N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), or 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffers. We examined the inhibitory effect on the interfering action of various reagents. The reagents examined were classified into two groups: a weak interference and a strong interference group. The interference of the strong interference group was suppressed by adding endotoxin and the test factors to LAL individually. Endotoxin peaks analyzed by gel-filtration HPLC disappeared in the presence of interfering factors. When buffers were used to prepare sample solutions instead of water, endotoxin peaks were maintained and interference with LAL reaction was suppressed. These results indicate that for the strong interference group, interference of the LAL reaction was a direct consequence of interfering factors binding to endotoxin. This alters endotoxin complexation, but this effect may be suppressed by preparing solutions in saline or other buffers instead of in water.     

Bacterial endotoxin testing: a report on the methods, background, data, and regulatory history of extraction recovery efficiency

Since the mid-1970s the Limulus Amebocyte Lysate (LAL) assay has been used to test medical devices for bacterial endotoxins. The Association for the Advancement of Medical Instrumentation (AAMI) recently published a standard designated ANSI/AAMI ST 72: 2002, Bacterial Endotoxins--Test methodologies, routine monitoring, and alternatives to batch testing, which addresses LAL testing and associated issues. In order to perform the bacterial endotoxins test (BET), the test article must be extracted in an aqueous medium, with the extract being used as the test solution. In the early years of testing, and periodically throughout LAL test history, questions have arisen about validation of the extraction efficiency of endotoxins from medical devices. The AAMI Microbiological Methods Committee appointed a Task Group to thoroughly research the issue of extraction efficiency and to recommend whether validation of extraction efficiency is necessary for LAL testing of medical devices.     

抗淋巴细胞免疫球蛋白制品中热原处理

对热原不合格的抗淋巴细胞免疫球蛋白 (ALG)半制品再制条件进行了研究。将高热原的ALG半制品通过使用碱、酸以及A5 0处理后 ,细菌内毒素的含量明显降低 ,热原质经家兔和鲎试剂检测均合格 ,其生物学效价仍不低于 1 :4 0 0 0 ,而且它们的其它各项主要指标也都达到中国生物制品暂行规程的要求。这一试验结果表明碱、酸处理并辅以A5 0吸附是去除ALG制品中热原的一种有效方法     

A quantitative in vitro assay method for detecting biological activity of endotoxin using prostaglandin E2 induction in rabbit peripheral blood

The pyrogen test and the endotoxin test (the LAL test) have been playing crucial roles in detecting endotoxin in parenteral drugs. The current test methods, however, have disadvantages such as requiring a large number of animals or an inadequacy in evaluation of in vivo endotoxin activity. We attempted to establish a new assay method that can overcome the shortcomings of the current methods. We standardized the in vitro assay method by the use of prostaglandin E2 (PGE2) induction from peripheral blood of rabbits for detecting endotoxin activity. A linear dose-response regression was attained from approximately 0.15 to 5 endotoxin units/ml of Japanese national reference standard endotoxin by the in vitro assay. The assay showed a fine correlation with the pyrogen test but not with the LAL test, when endotoxins from various bacterial sources were tested. The in vitro assay was also shown to have the capability of detecting a synergistic effect of endotoxin and parenteral drugs. The in vitro PGE2 induction test using rabbit blood was, therefore, suggested to be the appropriate test method for guaranteeing the same level of safety of parenteral drugs as the pyrogen test does.     

Use of magnesium to increase sensitivity of Limulus amoebocyte lysate for detection of endotoxin.

Increased sensitivity of the Limulus amoebocyte lysate (LAL) assay for the detection of endotoxin was attained by the reconstitution of commercially available LAL reagent with a magnesium-containing solution. As little as 2 to 6 pg (0.002 to 0.006 ng) of Escherichia coli O127:B8 endotoxin per ml was detected, an increase in sensitivity of 10 to 30 times. The optimum magnesium concentration range for the LAL reagent used and the optimum pH range were approximately 50 to 65 mM and pH 6.0 to 8.0, respectively. Reconstitution of five commercially available brands of LAL with a solution containing magnesium resulted in greater assay sensitivity than the identical LAL reconstituted with pyrogen-free water. Use of LAL reconstituted with a solution containing magnesium is crucial for the assay of some parenteral products, wherein increased sensitivity is essential to meet the requirement for the maximum valid dilution criteria. The mode of action of magnesium for enhanced sensitivity of LAL has been postulated.