细菌内毒素定量检测的影响因素分析及对策

细菌内毒素检查法(简称鲎试验)是利用鲎血变形细胞制成的鲎试剂与微量内毒素产生凝集反应的现象,作为判断样品中细菌内毒素含量是否符合规定的一种方法。其基本原理是利用微量内毒素激活、活化鲎试剂中的C因子和B因子,使凝固酶原转化为凝固酶,成为凝胶样的凝固蛋白,具有简便、迅速、特异性高及灵敏度高等优点。中国药典1995年板已收载,并对鲎试剂、内毒素标准、试验程序、判断标准等作了规定。 我科引进的EDS-99细菌内毒素检测系统可进行动态比浊法和显色基质法检测内毒素,分别利用细菌内毒素与鲎试剂形成凝胶过程中的浊度变化以及反应过程中产生的凝固酶使特     

两种方法测定灯盏细辛注射液中细菌内毒素含量

目的：建立灯盏细辛注射液细菌内毒素的定性和定量检测方法。方法：按照《中国药典》2010年版一部附录细菌内毒素检查法项下凝胶法和动态浊度法对灯盏细辛注射液进行试验。结果：灯盏细辛注射液注射液稀释25倍（凝胶法）和200倍（动态浊度法）后对鲎试剂及内毒素反应无干扰作用。结论：用凝胶法和动态浊度法检测灯盏细辛注射液中细菌内毒素的含量是可行的。     

鲎试验测定复方氨基酸(15)双肽(2)注射液中的细菌内毒素

目的:检测复方氨基酸(15)双肽(2)注射液中的细菌内毒素。方法:供试品干扰试验后,采用凝胶鲎试验对样品中的细菌内毒素进行检测。结果:三批复方氨基酸(15)双肽(2)注射液稀释至28倍时对细菌内毒素的检测无干扰作用,所测样品的细菌内毒素含量均小于7 EU.ml-1。结论:所建立方法检查复方氨基酸(15)双肽(2)注射液中的细菌内毒素可行。     

鲎试验测定长链脂肪乳注射液中细菌内毒素的方法学研究

目的：确定长链脂肪乳注射液中细菌内毒素鲎试验检查法的可行性。方法：供试品干扰试验后，采用凝胶鲎试验对样品中细菌内毒素进行检测。结果：三批长链脂肪乳注射液稀释至2倍时对细菌内毒素的检测无干扰作用，所测样品的细菌内毒素含量均小于0．5EU·ml^-1。结论：所建立方法检查长链脂肪注射液中的细菌内毒素可行。     

鲎试验测定注射用头孢雷特中细菌内毒素的方法学研究

目的：检测注射用头孢雷特中的细菌内毒素。方法：供试品干扰试验后,采用凝胶鲎试验对样品中细菌内毒素进行检测。结果：三批注射用头孢雷特浓度为2mg·ml^-1对细菌内毒素的检测无干扰作用,所测样品的头孢雷特细菌内毒素含量均小于0.5EU.mg-1。结论：所建立方法检查注射用头孢雷特中的细菌内毒素可行。     

动态比浊法测定13N-氨水的细菌内毒素含量

目的建立快速准确测定PET示踪剂13N-氨水中的细菌内毒素含量的方法.评估动态比浊法在半衰期短的PET临床用药中的实用价值. 方法通过制备5.0 Eu/ml、0.5 Eu/ml、0.05 Eu/ml的标准内毒素溶液进行测定,制出LogT-LogC 标准曲线图,根据样品的反应时间代入曲线方程得出13N-氨水中细菌内毒素的含量. 结果标准曲线在标准内毒素浓度范围为0.05Eu/ml～5.0Eu/ml时线性关系良好(γ=0.99969),精密度和样品阳性对照的回收率都比较满意.样品阳性对照的回收率一般为75%～120%,阳性样品回收率越接近100%,则表明此次检验的准确性越高.此方法最低检测范围为0.05Eu/ml. 结论与传统内毒素检测方法凝胶法相比,动态比浊法具有快速、准确、有效、定量地测定13N-氨水中的细菌内毒素含量的优势.     

双抗体夹心ELISA定量检测内毒素的方法学研究

目的 建立检测细菌内毒素（endotoxin,ET)的双抗体夹心ELISA法。方法 用自制的抗LPS－mAb C3A2和H3D3，分别作为包被和酶标记mAb，建立双抗体夹心ELISA法，并进行了实验条件的选择。然后用优化后的双抗夹心法对检测LPS的敏感性、特异性、重复性和回收率进行鉴定试验，并与鲎试验定量仪器比浊法和鲎试验定性法进行比较。结果 双抗体夹心法检测LPS的敏感性为50ng/L，特异性与准确性均高于仪器比浊法和鲎试验定性法，回收率是86.7%-97.4%，CV值为0.62%-4.8%。结论 建立的双抗夹心法，检测LPS的敏感性、准确性和特异性均较良好，为临床诊治内毒素血症提供了一种简便快速准确特异的实验工具。     

动态比浊法测定^13N—氨水的细菌内毒素含量

目的：建立快速准确测定PET示踪剂^13N-氨水中的细菌内毒素含量的方法。评估动态比浊法在半衰期短的PET临床用药中的实用价值。方法：通过制备5．0Eu/ml、0．5Eu/ml、0．05Eu/ml的标准内毒素溶液进行测定，制出LogT-LogC标准曲线图，根据样品的反应时间代入曲线方程得出^13N－氨水中细菌内毒素的含量。结果：标准曲线在标准内毒素浓度范围为0．05Eu/ml-5.0Eu/ml时线性关系良好（γ＝0．99969),精密度和样品阳性对照的回收率都比较满意。样品阳性对照的回收率一般为75％－120％，阳性样品回收率越接近100％，则表明此次检验的准确性越高。此方法最低检测范围为0．05Eu/ml。结论：与传统内毒素检测方法凝胶法相比，动态比浊法具有快速、准确、有效、定量地测定^13N－氨水中的细菌内毒素含量的优势。     

阳离子多肽MP-1对脓毒症模型小鼠的保护作用及机制

目的：观察阳离子多肽MP-1的体内外抗内毒素(LPS)活性并探讨其对脓毒症小鼠的保护机制。方法：采用LPS(20mg／kg)静脉注射攻击小鼠，观察静脉注射MP-1(3mg／kg)对小鼠的保护作用；应用生物传感器检测MP-1与LPS结合的亲合力，通过动态比浊法鲎试验定量检测MP-1对LPS的中和作用，并用反转录聚合酶链反应(RT-PCR)观察MP-1对LPS(100ng/ml)刺激的小鼠腹腔巨噬细胞(PMψ)TLR4、     

动态浊度法在血站细菌内毒素检测中的应用

目的探讨32孔动态浊度试管仪在血站细菌内毒素检测中的应用情况。方法对血站原辅材料进行抽检,使用动态浊度试管仪测定值做标准曲线,并检测其细菌内毒素含量。结果标准曲线显示该方法线性关系好,实验稳定,灵敏度高;原辅材料的细菌内毒素含量均小于药典的规定限,结果合格。结论 32孔动态浊度试管仪在血站原辅材料细菌内毒素检测中的应用良好,使用的原辅材料质量优良。     

Quantification of cultivable bacteria and endotoxin in post-treatment apical periodontitis before and after chemo-mechanical preparation

This clinical study was conducted to quantify cultivable bacteria and endotoxin in root canals with post-treatment apical periodontitis by correlating their levels with clinical features and to evaluate the effect of chemo-mechanical preparation (CMP) with 2 % chlorhexidine gel?+?17 % EDTA on bacterial and endotoxin removal/elimination. Moreover, target strict Gram-negative anaerobic bacteria were investigated by polymerase chain reaction (PCR). Fifteen teeth with post-treatment apical periodontitis were sampled before (s1) and after (s2) CMP. Culture techniques determined the number of colony-forming units (CFU). PCR (16S rDNA) and limulus amebocyte lysate (LAL) assay were used for bacterial and endotoxin detection, respectively. Prevotella nigrescens (4/15), Prevotella intermedia (2/15), and Tannerella forsythia (2/15) were the most frequently detected species. Endotoxin was recovered in 100 % of the samples. At s1, bacteria and endotoxin were detected at a median value of 5.14 × 10(3)?CFU/mL and 3.96 EU/mL, respectively. Higher levels of endotoxin were related to a larger size of radiolucent area (>5?mm) (p?相似文献   

Physical and biological properties of U.S. standard endotoxin EC after exposure to ionizing radiation.

Techniques that reduce the toxicity of bacterial endotoxins are useful for studying the relationship between structure and biological activity. We used ionizing radiation to detoxify a highly refined endotoxin preparation. U.S. standard endotoxin EC. Dose-dependent changes occurred by exposure to 60Co-radiation in the physical properties and biological activities of the endotoxin. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis showed gradual loss of the polysaccharide components (O-side chain and R-core) from the endotoxin molecules. In contrast, although endotoxin revealed a complex absorption pattern in the UV range, radiation treatment failed to modify that pattern. Dose-related destruction of the primary toxic component, lipid A, was suggested by the results of activity tests: both the pyrogenicity and limulus reactivity of the endotoxin were destroyed by increasing doses of radiation. The results indicate that the detoxification is probably due to multiple effects of the ionizing radiation on bacterial lipopolysaccharides, and the action involves (i) the destruction of polysaccharide moieties and possibly (ii) the alteration of lipid A component of the endotoxin molecule.     

Clearance of endotoxin from blood of rabbits injected with staphylococcal toxic shock syndrome toxin-1.

This study was undertaken to examine some of the properties of staphylococcal toxic shock syndrome toxin 1 (TSST-1) with regard to the clearance of endotoxin from blood. The concentration of endotoxin in blood was measured by using a chromogenic limulus test and modified perchloric acid method. When TSST-1, which produces fever in rabbits, was injected (100 ng/ml or 100 micrograms/ml per kg) intravenously (i.v.) into the animals, no measurable level of endotoxin was detected in the blood. In control animals, which were given 5 micrograms of endotoxin per ml per kg i.v., endotoxin could be detected in the blood at rapidly declining levels. These results suggested that TSST-1 might not lead bacterial endotoxin from other body sites into the blood. When the animals were given TSST-1 (1 to 100 ng/ml per kg) i.v. and then endotoxin (5 micrograms/ml per kg) i.v. 4 h later, endotoxin was detected in the blood at a high level, depending on the dose of TSST-1 injected. These results showed that TSST-1 inhibited the clearance of endotoxin in the blood; this clearance is thought to be mainly done by the reticuloendothelial system. In the animals given TSST-1 (100 ng/ml per kg) and endotoxin (5 micrograms/ml per kg) simultaneously, the endotoxin level in the blood was found to be higher than that in control animals given endotoxin only but lower than that in the animals given TSST-1 and then endotoxin at the same doses.     

两种生物材料细菌内毒素检查和热原检查方法比较的研究

目的本试验对样品管腔支架(镍钛)和支架输送系统进行热原检测.方法采用细菌内毒素检查和家兔检查法.结果细菌内毒素试验表明,两样品不合格;热原试验表明,两样品合格.结论导致鲎试剂凝集不一定就能引起生物体的热原反应.出现这种不相一致结果的原因很多.所以在评价生物材料和医疗器械时,建议做热原试验.     

慢性丙型肝炎患者血浆内毒素和血清MBL变化及临床意义

目的:探讨了慢性丙型肝炎患者血浆内毒素和血清甘露聚糖结合凝集素(MBL)水平的变化及意义.方法:应用鲎试验和酶联法对31例慢性丙型肝炎患者进行了血浆内毒素和血清MBL测定,并与35名正常健康人作比较.结果:慢性丙型肝炎患者血浆内毒素和血清MBL水平均非常显著地高于正常人水平(P<0.01),且内毒素水平与MBL呈正相关...     

Endotoxin release from Escherichia coli after exposure to tobramycin: dose-dependency and reduction in cefuroxime-induced endotoxin release

ObjectiveTo study the release of free endotoxin from Escherichia coli exposed to varying concentrations of the penicillin-binding protein (PBP) 3-specific β-lactam antibiotic cefuroxime, the aminoglycoside tobramycin, and a combination of the two, and to test the relationship between bacterial killing rate and endotoxin release.MethodsA clinical isolate of Escherichia coli in logarithmic phase was exposed to 0.1, 2, 10, and 50 × minimum inhibitory concentration (MIC) of cefuroxime, tobramycin, and a combination of the two. Samples for viable counts and endotoxin analysis were drawn immediately before and after the addition of the antibiotics and at 1, 2, 4, 6, and 24 h. All experiments were performed in triplicate. For the analysis of endotoxin, a chromogenic limulus amoebocyte lysate assay was used.ResultsEndotoxin liberation was found to be proportional to the number of killed bacteria for each antibiotic regimen at each concentration level justifying the endotoxin-liberating potential to be expressed as release of endotoxin per killed bacterium, an expression that was independent of the inoculum size. At all concentration levels there was a statistically significant difference between the treatments, with the highest release of endotoxin per killed bacterium for cefuroxime, lower for tobramycin and the lowest for the combination of the two drugs (P < 0.001). With increasing doses, there was a significant reduction (P < 0.001) in the propensity to release endotoxin. When the bacterial killing rate was correlated to the propensity to release endotoxin in bacteria exposed to tobramycin or the combination of tobramycin and cefuroxime, a significant negative correlation was found (P < 0.01). This reduction in endotoxin release was not caused by an unspecific endotoxin binding of tobramycin.ConclusionsAddition of tobramycin reduced the cefuroxime-induced endotoxin release per killed bacterium to a level which was even lower than that of tobramycin alone in spite of an increased killing rate. Increasing concentrations of tobramycin led to reduction in endotoxin release, which may be of benefit when dosing aminoglycosides once daily.     

重症胰腺炎大鼠肠道免疫功能改变及精氨酸的调节作用

目的：探讨重症急性胰腺炎（Severe Acute Pancreatitis，SAP）大鼠肠道黏膜免疫功能变化及L-精氨酸（L-Arg）对SAP大鼠肠道黏膜免疫功能的影响。方法：成年、雄性Wistar大鼠随机分为胰腺炎组、假手术组和L．精氨酸治疗组。分别于造模后24、48和72小时取标本检测：鲎试剂法检测大鼠门静脉血内毒索水平、免疫组化方法检测并计数小肠黏膜固有层内CD3、CD4、CD8阳性T淋巴细胞百分数、放射免疫法检测盲肠内容物中分泌型IgA（sIgA）含量。结果：SAP组大鼠各时段门静脉血内毒素水平显著升高，回肠末段黏膜固有层CD3^＋、CD4^＋T淋巴细胞百分数明显减少，CD^＋／CD8^＋T淋巴细胞比值降低，盲肠内容物sIgA含量明显降低；与SAP组比较，L-精氨酸组大鼠各时段门静脉血内毒素水平明显降低，回肠末段黏膜固有层CD3^＋、CD4^＋T淋巴细胞百分数明显增加，CD4^＋／CD8^＋T淋巴细胞数比值升高，盲肠内容物SIgA含量增加。结论：SAP大鼠早期即发生肠黏膜免疫功能明显下降，可能是导致肠道内毒素移位的主要原因，L-精氨酸可以改善SAP大鼠肠道黏膜免疫功能，降低SAP大鼠肠道内毒索移位发生。     

Statistical determination of endotoxin content in influenza virus vaccine by the limulus amoebocyte lysate test.

To determine laboratory-to-laboratory variability of the limulus amoebocyte lysate (LAL) test for evaluating endotoxin content in influenza virus vaccine, a collaborative study was designed. Participants were six influenza virus vaccine manufacturers and the Bureau of Biologics (BoB). Lysate lot 116, Reference Influenza Virus Vaccine for Endotoxin Assay E-1, and four test vaccines having different ratios of LAL activity relative to reference E-1 were supplied by the BoB. Each laboratory used its normal test procedure. All vaccines were coded. One pair (reference and test) of vaccines per day was tested for 4 days a week over a 4-week period. All data were analyzed at the BoB. The degree of variability experienced by testing laboratories was estimated by the study. This estimate did not conflict with experience gained from previous routine testing in any of the participating laboratories. A statistical approach to the evaluation of LAL data from testing influenza virus vaccine for endotoxin content was developed based upon the overall variation obtained from the collaborative study.     

Chemical and biological evaluation of endotoxin contamination on natural rubber latex products

Relationship between pyrogenicity and bacterial endotoxin contamination on latex products was demonstrated by chemical analysis and biological assays. In commercially available latex products' surveillance, water extracts prepared from one surgical glove and two silicone elastomer-coated Foley catheters sterilized by gamma-irradiation were obviously pyrogenic in rabbits. The induced fever was monophasic at low dose of the pyrogenic extracts and biphasic at high dose. These extracts exhibited limulus amebocyte lysate gelation activity, and induced inflammatory cytokine (interleukin-1, interleukin-6, and tumor necrosis factor-alpha) production from MM6-CA8 human monocytoid cells. These biological properties, including pyrogenicity, completely disappeared by treating the pyrogenic extracts with endotoxin-adsorbent affinity column. Limulus amebocyte lysate activity and cytokine production from MM6-CA8 cells induced by the extracts were significantly decreased by endotoxin inhibitors, an active fragment peptide of an 18-kDa cationic antimicrobial protein and a synthetic lipid A B464 analogue. Furthermore, very small amounts of 2-keto-3-deoxyoctonate and 3-hydroxy fatty acid, which are common constituents of bacterial endotoxins, were detected by gas chromatography-mass spectrometry analysis of the pyrogenic extracts. These findings clearly showed that the pyrogenicity found in these latex products originated from endotoxins contaminating the products.