动态比浊法鲎试验定量分析催化剂的细菌内毒素

目的通过定量检测催化剂的细菌内毒素含量,为控制药品质量及筛选最佳生产工艺提供参考。方法应用动态比浊法及凝胶法鲎试验对催化剂的细菌内毒素进行定量和半定量检测。结果动态比浊法鲎试验定量检测了4批催化剂,其原液(原瓶加5.0mL水)细菌内毒素含量依次为20EumL-1-50EumL-1不等,样品在256倍稀释时其添加内毒素(2.0EumL-1)的回收率在75%-125%之间,此时对鲎试验反应无干扰作用,与凝胶法的检查结果一致。结论动态比浊法鲎试验可用来定量检测催化剂的细菌内毒素含量。     

槲皮素、松萝酸对伴放线放线杆菌产生内毒素的抑制作用研究

目的 研究不同浓度下槲皮素、松萝酸对伴放线放线杆菌产生内毒素的抑制作用.方法 采用显色基质鲎试验法检测不同浓度药物作用下的伴放线放线杆菌菌液中留存的内毒素量.结果 槲皮素组与松萝酸组中伴放线放线杆菌菌液产生内毒素的量明显低于阴性对照组.经检验,t(谢)N =3.890,t松N =6.120,P值均＜0.01,具有显著统计学差异.结论 槲皮素与松萝酸对伴放线放线杆菌产生内毒素均有抑制作用,作用和效果与药物浓度呈正相关性,松萝酸的抑制作用强于槲皮素.     

家兔法与内毒素法检查生物材料热原的适用性研究

药品与生物材料在生物安全性评价方法上有着很大的区别。临床上广泛运用内毒素法检查药品热原,然而,运用细菌内毒素法进行部分组成成分较为复杂的生物材料的热原试验是否适当有待明确。本研究在2005版药典的基础上,分别运用内毒素法和家兔法对两种组织工程支架材料进行热原试验的比较研究,实验结果表明运用内毒素法得到的试验结果为阴性,运用家兔法得到的试验结果为阳性。这两种方法分别测定每种材料所得到的热原试验结果不相符合,表明对组成成分复杂的生物材料,含热原的因素较为复杂,用家兔法进行试验检测热原可能更加灵敏。     

生物传感技术应用于中和内毒素活性物质的研究

目的 :探讨应用生物亲合传感技术测定中和内毒素活性物质。方法 :多粘菌素B与一定浓度的内毒素混合后 ,分别用生物亲合传感技术和鲎试验法 ,测定多粘菌素B中和内毒素的比值 ,同时用生物学方法对结果进行进一步验证。结果 :多粘菌素B中和内毒素的比值 ,生物传感器技术为 0 .3 5ug∶1ng、动态浊度法为 0 .5mg∶1、基质显色法为 1mg∶1 ,生物学测定结果与生物亲合传感技术测定结果相同。结论 :运用生物亲合传感技术测定中和内毒素活性物质的结果更为准确、方便、快速 ,值得进一步推广应用生物传感技术应用于中和内毒素活性物质的研究@吕根法$…     

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。     

家兔法与内毒素法检查生物材料热原的适用性研究

药品与生物材料在生物安全性评价方法上有着很大的区别.临床上广泛运用内毒素法检查药品热原,然而,运用细菌内毒素法进行部分组成成分较为复杂的生物材料的热原试验是否适当有待明确.本研究在2005版药典的基础上,分别运用内毒素法和家兔法对两种组织工程支架材料进行热原试验的比较研究,实验结果表明运用内毒素法得到的试验结果为阴性,...     

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     

慢性重型肝炎患者血中LPS、sCD14变化的临床意义

我们检测 2 4例慢性重型肝炎 (慢重肝 )患者血中内毒素(ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ ,ＬＰＳ)及可溶性ＣＤ14(ｓＣＤ14)的水平 ,现将结果报告如下。1　材料和方法1 1　检测对象　临床确诊慢重肝患者 2 4例 (分期例数见表1) ,健康献血员对照组 10例。慢性乙型肝炎 (慢乙肝 )患者16例。1 2　检查方法　内毒素的测定采用基质显色法 ,试剂盒购自上海伊华临床医学科技公司 ,血浆内毒素浓度≥ 5 0ｐｇ/ｍｌ定为内毒素血症 (ＥＴＭ)。ｓＣＤ14和肿瘤坏死因子 (ＴＮＦ α)的测定应用ＥＬＩＳＡ双抗体夹心法 ,试剂盒均由深圳晶美生物工程有…     

光纤间接免疫荧光法检测肺炎支原体抗体

目的和方法:首次采用自行设计、研制的光纤荧光光度计对肺炎支原体抗体进行了检测,并将该法与临床上现行ELISA法及荧光显微镜法进行比较测定.结果:此法标准曲线的相关系数r=0.9930,测定的相对荧光强度(△F)大于2.0为阳性,小于2.0为阴性,且稳定性好.检测35人次临床血清标本,18例阳性标本中,本法有19例为阳性,与ELISA法及荧光显微镜法符合率(特异性)为94.4%;17例阴性标本中,本法有16例为阴性,符合率为94%结论:表明本光纤法具有特异性好、敏感性高,还具有可定量,操作简便快速,所需样本量微,所用仪器新颖、价廉、易普及等优点.     

脱细胞粘膜基质的热原试验研究

目的 考察脱细胞粘膜基质的热原试验方法的可行性。方法 依据《中华人民共和国药典》2010年版二部附录XIE细菌内毒素检查法和ISO10993.12 Biological evaluation of medical devices-Part 12:Sample preparation and reference materials,使用离心法消除样品浸提液的悬浮颗粒,使用动态浊度法验证离心条件是否会影响浸提液中细菌内毒素的检测。依据《中华人民共和国药典》2010年版三部附录XIID热原检查法检测脱细胞粘膜基质的热原试验是否合格。结果 本实验中采用的离心条件不会影响样品浸提液中细菌内毒素的检测,热原试验合格。结论该产品可以采用此离心条件进行热原检测。     

Methodological and clinical evaluation of a microplate assay of endotoxin in whole blood sample

We have developed a simple, rapid method for determining endotoxin in whole blood using a microtiter plate. The samples were pretreated with 0.66 mol/l nitric acid containing 0.25% Triton X-100; the supernatants were placed in a microtiter plate well, and incubated with endotoxin-specific chromogenic limulus test reagent (Endospecy; Seikagaku Corporation). The calibration curve was linear from 0 to 50 pg/ml of Escherichia coli 0111: B4 endotoxin with a variation coefficient less than 5%. The recovery of endotoxin added to human and experimental animals was about 100% without being affected by dilution. The normal endotoxin level in human whole blood was less than 10 pg/ml in reference to E. coli 0111: B4 endotoxin. The results in 120 patients with various diseases correlated well with those determined by a conventional method using plasma as a sample (r = 0.877; p less than 0.05). Whole blood sample has advantages over plasma in that it allows all endotoxin in a sample to be determined, requires a smaller amount of sample, and has less risk of contamination.     

Endotoxin exposure, wheezing, and rash in infancy in a New Zealand birth cohort

BACKGROUND: Wheezing in infancy is common and is associated with small lungs, viral respiratory tract infection, and environmental tobacco smoke exposure. Recently, increased levels of endotoxin in the domestic environment have also been associated with infant wheezing, particularly among infants with a family history of atopic disease. OBJECTIVE: To explore associations between exposure to endotoxin at 3 months of age and reported symptoms of wheezing, rhinitis, itchy scaly rash, and atopy at 15 months in a birth cohort of 881 New Zealand children. METHODS: Using standardized methods, a 1-m(2) site from the bedroom floors of the 3-month-old infants was sampled and analyzed for endotoxin. RESULTS: Wheezing was significantly associated with higher endotoxin levels (odds ratio [OR], 1.54; 95% CI, 1.03-2.30), particularly among infants with a parental history of allergic disease (OR, 1.67; 95% CI, 1.07-2.60). Higher endotoxin concentrations were also strongly associated with recurrent itchy rashes (OR, 1.87; 95% CI, 1.14-3.05), particularly among infants who were atopic (OR, 4.64; 95% CI, 1.56-13.77) or had a parental history of allergic disease (OR, 2.10; 95% CI, 1.22-3.61). CONCLUSION: Domestic endotoxin was associated with reported airway and skin symptoms in this large group of New Zealand infants. The role of endotoxin in the development of respiratory and skin disease in infancy deserves further study. CLINICAL IMPLICATIONS: Reducing domestic endotoxin exposure might reduce infant wheezing and atopic dermatitis, but the long-term benefits of this remain unclear.     

Enhancement of host susceptibility to lethal endotoxin shock by staphylococcal pyrogenic exotoxin type C.

Staphylococcal pyrogenic exotoxin (PE) ty pe C enhanced the susceptibility of rabbits to lethal shock by endotoxin by as much as 50,000-fold. A graph of log PE type C dose used for pretreatment versus log 50% lethal dose of endotoxin gave a straight line with a slope of approximately -1. Rabbits that received PE type C alone showed fevers only, but those given both PE ty pe C and endotoxin showed initial fever followed by hypothermia, labored breathing, diarrhea, evidence of vascular collapse, and finally death. When a PE type C dose of 3 micrograms/kg was used, pretreatment of the animals with PE for 2 h before giving the endotoxin was required to obtain maximal susceptibility. However, when 15 micrograms of PE type C per kg was utilized, the endotoxin could be given before, concurrently, or after PE type C. The capacity of PE type C to prepare rabbits for enhanced susceptibility to endotoxin was lost after 24 to 48 h. Animals could be protected from enhanced susceptibility to endotoxin by prior immunization with either PE type C or endotoxin. However, 30% of the rabbits which were immunized with PE type C failed to develop immunity, and after three injections of PE type C, these animals developed gram-negative bacteremia and succumbed. In addition, rabbits with diarrhea initially, possibly caused by Pasteurella infection, died less than 24 h after a single injection of PE type C.     

Exposure to house dust endotoxin and allergic sensitization in adults

BACKGROUND: It has been suggested that exposure to elevated levels of endotoxin decreases the risk of allergic sensitization. OBJECTIVE: To examine the associations between current exposure to bacterial endotoxin in house dust and allergic sensitization in adults. METHODS: In 1995-1996, we conducted a nested case-control study following a cross-sectional study performed within the European Community Respiratory Health Survey (ECRHS). Data of 350 adults aged 25-50 years was analysed. Allergic sensitization was assessed by measurement of specific immunoglobulin E (IgE) against several inhalant allergens. Living room floor dust samples were taken. The endotoxin content was quantified using a chromogenic kinetic Limulus amoebocyte lysate test. RESULTS: Multiple logistic regression analysis showed a negative association between exposure to house dust endotoxin and severe allergic sensitization. Odds ratios (95% CI) adjusted for place of residence, gender, age, and 'caseness' were 0.80 (0.64-1.00) for sensitization to >/=1 allergen and 0.72 (0.56, 0.92) for sensitization to >/=2 allergens using 3.5 kU/l as a cut-off value for sensitization. With regard to single allergens, the protective effect of endotoxin was strongest for pollen sensitization [aOR (95% CI) = 0.74 (0.58, 0.93)]. CONCLUSION: Our results indicate that current exposure to higher levels of house dust endotoxin might be associated with a decreased odds of allergic sensitization in adults.     

Direct induction of tissue factor synthesis by endotoxin in human macrophages from diverse anatomical sites.

On exposure to endotoxin and other stimuli, human peripheral-blood mononuclear cells generate a potent procoagulant activity (PCA), identified as tissue factor. Although it is now recognized that the monocytes are the source of PCA, the question whether these cells per se are capable of procoagulant response to endotoxin or require lymphocyte collaboration remains unsettled. We have investigated the capacity of highly purified human macrophages from diverse anatomical sites to generate PCA following endotoxin stimulation. Purified (greater than 99%) monocyte-derived macrophages were obtained by prolonged (3-10 days) in-vitro culture of adherent monocytes using medium supplemented with 50% human serum. Purified (greater than 95%) peritoneal and milk macrophages were isolated by adherence to plastic. PCA was measured before and after incubation (4 hr at 37 degrees) with endotoxin (Salmonella enteritidis LPS, W or Escherichia coli O111:B4LPS, W, 1 microgram/ml final concentration) using a one-stage clotting assay and/or a two-stage amidolytic assay. Monocyte-derived macrophages had low baseline PCA (14-19 units/10(5) cells) but, upon exposure to endotoxin, displayed an eight-fold increase in PCA over control. Peritoneal and milk macrophages expressed very low baseline activity (1-5 units/10(5) cells). The latter, however, increased 15-20 times over control following endotoxin stimulation. PCA was identified as tissue factor by biological and immunological criteria. Its generation was completely abolished by cycloheximide. It is concluded that in the human mononuclear phagocyte series the capacity to produce PCA is not restricted to circulating monocytes but is also expressed by macrophages obtained from diverse anatomical sites. These macrophages appear to be autonomous in their procoagulant response to endotoxin.     

Exposure to pets, and the association with hay fever, asthma, and atopic sensitization in rural children

BACKGROUND: An increasing number of studies report pet exposure to be associated with lower risk of asthma and allergies. This 'protective pet effect' has been suggested to result from a modified T-helper (Th)2-cell response, or because of increased microbial load in homes where pets are kept. We examined the associations between pet contact and the occurrence of asthma and allergies in children of the rural Allergy and Endotoxin (ALEX) population, taking farm animal contact, endotoxin and cat allergen levels in mattress dust into account. METHODS: Information about contact with pets and farm animals, asthma and allergy were collected for 812 children by a standardized parents' questionnaire and an interview. Mattress dust endotoxin and cat allergen levels as well as specific IgE and IgG4 antibodies to Fel d1 were determined. RESULTS: Current contact with dogs was inversely associated with diagnosed hay fever (OR 0.26, 95% CI 0.11-0.57), diagnosed asthma (OR 0.29, 95% CI 0.12-0.71), sensitization to cat allergen (OR 0.48, 95% CI 0.23-0.99) and to grass pollen (OR 0.55, 95% CI 0.33-0.94), but not with increased IgG4 levels. Early and current contact with cats were associated with reduced risk of wheezing (OR 0.48, 95% CI 0.23-1.00, and OR 0.49, 95% CI 0.26-0.92, respectively) and grass pollen sensitization. Adjustment for farm animal contact but not for endotoxin and cat allergen exposure attenuated these associations and the effect of pet was stronger among farmers' children. CONCLUSION: Although pet exposure was very frequent in this rural population, the inverse relation between current dog contact, asthma and allergy was mostly explained by simultaneously occurring exposure to stable animals or was restricted to farm children. In addition, a subtle form of pet avoidance may contribute to the protective effect of pet.     

Properties of binding of Escherichia coli endotoxin to various matrices.

Binding of Escherichia coli O127:B8 endotoxin to a variety of resins and column materials was investigated by measuring the beta-hydroxy myristic acid content (a major component of the lipid A moiety) of endotoxin after hydrolysis by selected ion-monitoring gas chromatography-mass spectrometry. More than 80% of the endotoxin was bound to hydroxylapatite, polystyrene, Dowex 1-X2, and charcoal. The binding of endotoxin to these materials was markedly reduced by the addition of normal or delipidated serum. Phenyl- and octyl-Sepharose bound 56 and 50% of the endotoxin from saline solutions, respectively. Their percent binding was increased significantly in 1 M ammonium sulfate solutions, indicating hydrophobic interactions between endotoxin and phenyl- and octyl-Sepharose. Only 5% of the endotoxin was bound to plastic polymer PSI-HAP-100 beads, and no binding was observed with concanavalin A- and heparin-Sepharose. Study of the in vitro binding of endotoxin to the above material in the presence of serum suggests that the use of these materials in removing circulating endotoxin in vivo is limited.     

The effect of surgical stress and endotoxin-induced sepsis on the NK-cell activity, distribution and pulmonary clearance of YAC-1 and melanoma cells

Following surgery the activity of natural killer (NK) cells is decreased in the blood. It is possible that sepsis with release of endotoxin will further decrease the NK-cell activity. The purpose of the present study was to investigate the NK-cell cytotoxicity, the clearance in the lungs of YAC-1 and melanoma cells, as well as the distribution of NK-cells in the liver, following abdominal surgery and intraperitoneally (i.p.) administered endotoxin. Ten mice in each group were allocated to abdominal surgery, i.p. endotoxin or anaesthesia alone. Following abdominal surgery, the cytotoxicity of NK-cells isolated from the spleen was decreased and 4 h after injection the clearance of YAC-1 cells from the lungs was only 79.5+/-6.1% compared to 99.5+/-0.3% in the control group. The number of NK-cells in the liver was also significantly reduced following abdominal surgery. In contrast, i.p. endotoxin increased the activity of NK-cells by 28.5% compared to 11.8% in the control group and 8.1% in the surgery group, lowered the number of melanoma metastases in extrapulmonary organs and significantly increased the number of NK-cells in the liver. Following abdominal surgery, activity of NK-cells, pulmonary clearance and number of NK-cells in the liver were decreased. The number of NK-cells in the liver correlated with the NK-cell activity throughout the study. The increased NK-cell cytotoxicity and the increased number of NK-cells in the liver following i.p. administered endotoxin might initially be an appropriate measure against intra-abdominal infection.     

D,L-聚乳酸基形状记忆聚合物的生物安全性和细胞相容性

背景：课题组前期实验研制了输卵管避孕器材料D, L-聚乳酸基形状记忆聚合物,依据国内《生物材料和医疗器材生物学评价技术要求》规定,植入体内的组织工程材料必须进行生物安全评价和细胞相容性实验。 目的：观察D, L-聚乳酸基形状记忆聚合物的生物安全性。 方法：①内毒素实验：在鲎试剂中分别加入聚合物浸提液、内毒素工作标准品溶液和细菌内毒素检查用水。②致敏实验：在昆明小鼠肩胛骨内侧分别注射聚合物浸提液+弗氏完全佐剂+生理盐水、弗氏完全佐剂+生理盐水,通过皮内诱导、局部诱导和激发阶段,观察动物激发部位皮肤红斑和水肿反应程度。③急性毒性实验：分别在昆明小鼠腹腔注射100%,50%,25%聚合物浸提液及生理盐水。④细胞增殖MTT实验：直接法为将人脐静脉内皮细胞分别接种于聚合物膜、聚乳酸与玻璃片上;间接法为将人脐静脉内皮细胞分别接种于聚合物浸提液、丙烯酰胺溶液及1640培养液。 结果与结论：D, L-聚乳酸基形状记忆聚合物材料无细菌污染状况,符合生物安全标准,无致敏性及毒性,并且具有较好的细胞相容性。     

基于叉指金电极和声表面波技术的细菌内毒素快速检测方法的研究

目的 建立一种可用于定量细菌内毒素浓度的快速检测方法.方法 通过在叉指金电极上固定γ-氨丙基三乙氧基硅烷(3-APTES)和戊二醛为载体的多粘菌素B(PMB),使其与细菌内毒素的生物活性物质脂多糖发生反应；检测装置采用基于声表面波技术的双路差分电路,2路输出振荡信号经过混频器进行差分,根据频移量的变化表征内毒素含量.结果 频移变化与内毒素含量在75～130 EU/ml范围内基本成线性关系.结论 与传统法相比,检测时间缩短到10 min,测试灵敏度达到13.8 kHz/(EU·ml-1),重复性达到90％以上.