重组人戊型肝炎疫苗细菌内毒素检查方法的建立

确立重组人戊型肝炎疫苗原液的细菌内毒素检查方法。供试品参照《中华人民共和国药典》(2005年版三部)热原检查法进行热原检查,结果符合规定。该供试品同时参照《中华人民共和国药典》(2005年版三部)细菌内毒素检查法要求进行试验。供试品溶液在40μg/m l浓度下,确定内毒素限值为40EU/m l。供试品在该内毒素限值下干扰试验有效,且细菌内毒素检查法符合规定。该疫苗用内毒素检查法代替热原检查法,方法可行。     

鲎试验法检测A群流脑多糖菌苗中内毒素的研究

本文应用鲎试验凝胶法定量测定了２１２批Ａ群流脑多糖菌苗中内毒素含量,并与家兔热原试验结果进行了比较,通过试验求得内毒素标准品对家兔体温升温变化关系的线性方程和内毒素对家兔最小致热剂量。结果表明,占检定总批数９４．３％的制品中内毒素含量＜１００ＩＵ／μｇ多糖,家兔热原试验合格率为１００％,５．７％的制品中内毒素含量≥１００ＩＵ／μｇ多糖,家兔热原试验合格率为５８．３％。根据实验结果,可制订用凝胶法替代家兔法进行Ａ群流脑多糖菌苗的热原质试验的判定标准。作者建议：内毒素含量＜１００ＩＵ／μｇ多糖者,热原质试验判为合格,≥１００ＩＵ／μｇ多糖者,用家兔法予以仲裁。     

细菌内毒素检测法在注射用肝复肽检测中的应用

通过对注射用肝复肽稀释后,采用细菌内毒素试验对其进行检测,并与家兔热原试验相比较。结果表明,细菌内毒素试验可以替代家兔热原试验用于注射用肝复肽的检测。     

浙江省热原检查用兔敏感性状况调查

目的调查浙江省热原检查用兔对细菌内毒素的敏感性情况,为提高热原检查结果的准确性和可靠性提供参考。方法对全省所有取得生产许可证单位的家兔,用国家颁发的细菌内毒素标准品,＂热原检查法＂进行检查,剂量分别为5EU/Kg和10EU/Kg,记录并比较各单位家兔的平均升温值和升温率。结果对细菌内毒素,各兔场家兔的敏感性有一定的差异。静脉注射5EU/kg,平均升温值为0.40℃～0.87℃,升温率为35%～83%;静脉注射10EU/kg,平均升温值为0.74℃～1.16℃,升温率为70%～94%。结论不同生产单位的家兔对细菌内毒素的敏感性不同,有必要对热原检查用家兔进行细菌内毒素敏感性检查。     

关于建立栀子提取物热原检测方法的考察

目的根据2015版《中国药典》规定的新品种建立热原的指导原则,建立栀子提取物热原的测定方法。方法用国家颁发的细菌内毒素标准品,对设限值剂量的供试品组、10EU/kg内毒素组与10EU/kg内毒素的供试品组同时进行热原检查试验,观察三组家兔的升温情况。结果以上三组同时进行热原检查试验,限值剂量的供试品组结果无异常反应并符合规定,10EU/kg内毒素组与10EU/kg内毒素的供试品组的家兔升温显著且结果为不符合规定。结论以上三组同时进行热原检查试验,限值剂量的供试品组无异常反应并符合规定,10EU/kg内毒素组与10EU/kg内毒素的供试品组的家兔升温显著且结果为不符合规定。说明用该方法对栀子提取物进行热原检查是有效的。     

基因治疗相关产品对热原检测提出的挑战

本文就基因治疗相关产品这一类新制品的热原检测问题进行了分析讨论。将现有的国家法规规定的家兔热原检查法和内毒素检查法对该类产品的检测中存在的缺陷进行了分析，提出了热原检测对该类制品检测时面对的三个挑战；并将国外正在研究、发展的一种新的体外热原检测方法的优势进行了介绍。     

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     

生化药物生产过程中控制热原的方法

热原是微生物的代谢产物 ,是微生物的一种内毒素 ,其主要成分是脂多糖 ,分子量一般为 10 6左右。去除热原的主要方法有 :1 高温法。多在 2 5 0℃加热 30分钟以上。2 酸碱法。用酸或碱进行处理 ,破坏热原。3 吸附法。常用 0 1%～ 0 5 %的活性炭吸附处理。4 层析法。利用离子交换树脂或分子筛凝胶层析处理。5 超滤法。使用超滤装置滤除热原物质。为了保证热原符合规定 ,往往要经过多次实验才能选择合适的方法 ,设计出适宜的...     

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 …     

鲎C因子的性质、结构、功能及应用

鲎C因子是一种分子量为123kD的丝氨酸蛋白酶原,主要由六种结构域构成,能特异地结合内毒素分子而被激活。在内毒素的检测,抗内毒素治疗,去除生物制品中污染的内毒素,以及在抗微生物作用中有多种潜在的应用价值 。     

Comparison of the effects of several endotoxin preparations on body temperature and metabolic rate in the rat

The effects of several bacterial endotoxins on body temperature and resting oxygen consumption (VO2) were compared in normal rats. Low doses (0.05 mg/kg, i.m.) of 0127:B8 phenol-extracted endotoxin caused significant increases in both parameters. Maximal febrile responses (+1.6 degrees C) occurred at a dose of 0.05 mg/kg, but higher doses produced smaller effects. The maximal increase in VO2 (17%) occurred at doses of 0.5-1.0 mg/kg. A TCA extract of the same strain of endotoxin elicited a similar pattern of responses but was less potent than the phenol extract, whereas another endotoxin 026:B6 (TCA extract) was much less potent. The data illustrate the importance of constructing dose-response curves when comparing different endotoxins and indicate that in the rat, oxygen consumption provides a useful index of the response to pyrogens.     

Determination of endotoxins in sugar with the Limulus test

The Limulus amebocyte lysate test has been used for determination of pyrogens in sugar of different qualities. All the samples of domestic white sugar and beet raw sugar produced in Sweden during 1976 had a very low content of endotoxins, less than 10 ng/g of sugar. Imported cane raw sugar was, however, highly contaminated. The highest value obtained corresponds to about 100 mg of Escherichia coli endotoxin per g of raw sugar. Such crude sugar cannot, even after refining, be used for medical purposes. Instead, Swedish beet sugar is used as the raw material for production of invert sugar solutions for parenteral administration. The amount of endotoxin in this sugar is less than 1 ng/g.     

Determination of endotoxins in sugar with the Limulus test.

The Limulus amebocyte lysate test has been used for determination of pyrogens in sugar of different qualities. All the samples of domestic white sugar and beet raw sugar produced in Sweden during 1976 had a very low content of endotoxins, less than 10 ng/g of sugar. Imported cane raw sugar was, however, highly contaminated. The highest value obtained corresponds to about 100 mg of Escherichia coli endotoxin per g of raw sugar. Such crude sugar cannot, even after refining, be used for medical purposes. Instead, Swedish beet sugar is used as the raw material for production of invert sugar solutions for parenteral administration. The amount of endotoxin in this sugar is less than 1 ng/g.     

Filtration removal of endotoxin (pyrogens) in solution in different states of aggregation.

Bacterial lipopolysaccharides are recognized as the major cause of pyrogenic reactions from parenteral solutions. Molecular filtration was used to remove these pyrogenic molecules (endotoxins) from contaminated parenteral solutions. Because bacterial lipopolysaccharides can exist in different states of aggregation, depending on the composition of the solution they are suspended in, the full range of possible states of aggregation was examined by using filters with a wide range of pore sizes. Filters of different pore sizes retained endotoxin lipopolysaccharide presumed to be in the vesicle form, the micelle form, or the detergent-solubilized form in aqueous solutions. Endotoxins (pyrogens) were successfully removed from artificially contaminated solutions of concentrated antibiotics by using filters of 10,000-nominal-molecular-weight limit.     

Filtration removal of endotoxin (pyrogens) in solution in different states of aggregation.

Bacterial lipopolysaccharides are recognized as the major cause of pyrogenic reactions from parenteral solutions. Molecular filtration was used to remove these pyrogenic molecules (endotoxins) from contaminated parenteral solutions. Because bacterial lipopolysaccharides can exist in different states of aggregation, depending on the composition of the solution they are suspended in, the full range of possible states of aggregation was examined by using filters with a wide range of pore sizes. Filters of different pore sizes retained endotoxin lipopolysaccharide presumed to be in the vesicle form, the micelle form, or the detergent-solubilized form in aqueous solutions. Endotoxins (pyrogens) were successfully removed from artificially contaminated solutions of concentrated antibiotics by using filters of 10,000-nominal-molecular-weight limit.     

Induction of fever in a teleost fish (Lepomis gibbosus, L.)

Although fishes are ectotherms they are nevertheless able to thermoregulate behaviorally by selecting appropriate water temperatures (1). In a temperature gradient fish will congregate to a species-specific range of preferred temperature (“final thermal referendum”) which is unaffected by previous thermal history of the individual (2,3). Several aquatic (and terrestrial) ectothermic vertebrates have been found to exhibit “behavioral fever” which is manifested as an increase in preferred temperature above the final thermal preferendum (4). Fever can be elicited by pyrogens: whole bacteria (alive or killed), components of bacterial cellwall (endotoxins), endogenous pyrogens, prostaglandins or from several other sources (5). Since the results with fever induction in fish using whole bacteria or endotoxins are very scarce the aim of the present work was to compare possible thermoregulatory effects of endotoxins and prostaglandins in the same species (Lepomis gibbosus, L.) by means of identical methods.     

Specific binding of endotoxin to human monocytes and mouse macrophages: serum requirement

Specific binding of Bordetella pertussis and Neisseria meningitidis endotoxins to human monocytes and murine macrophages was demonstrated. Binding of B. pertussis endotoxin could be inhibited by endotoxins of Salmonella minnesota, Escherichia coli, and Klebsiella pneumoniae, the extent of inhibition being dependent on the origin of the lipopolysaccharides and on the origin of the mononuclear phagocytic cells. The binding of B. pertussis and N. meningitidis endotoxins which was mediated by the polysaccharide region of the endotoxins was serum dependent. The results indicated that the binding of endotoxin was promoted neither by natural antibodies directed against the endotoxin nor by proteins known to combine with endotoxins: immunoglobulins, albumin, or fibronectin; we have provided some evidence that complement components may play a role in the specific binding of endotoxins to the monocyte/macrophage membrane.     

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.     

Molecular basis of reduced potency of underacylated endotoxins

Potent TLR4-dependent cell activation by gram-negative bacterial endotoxins depends on sequential endotoxin-protein and protein-protein interactions with LPS-binding protein, CD14, myeloid differentiation protein 2 (MD-2), and TLR4. Previous studies have suggested that reduced agonist potency of underacylated endotoxins (i.e., tetra- or penta- vs hexa-acylated) is determined by post-CD14 interactions. To better define the molecular basis of the differences in agonist potency of endotoxins differing in fatty acid acylation, we compared endotoxins (lipooligosaccharides (LOS)) from hexa-acylated wild-type (wt), penta-acylated mutant msbB meningococcal strains as well as tetra-acylated LOS generated by treatment of wt LOS with the deacylating enzyme, acyloxyacylhydrolase. To facilitate assay of endotoxin:protein and endotoxin:cell interactions, the endotoxins were purified after metabolic labeling with [3H]- or [14C]acetate. All LOS species tested formed monomeric complexes with MD-2 in an LPS-binding protein- and CD14-dependent manner with similar efficiency. However, msbB LOS:MD-2 and acyloxyacylhydrolase-treated LOS:MD-2 were at least 10-fold less potent in inducing TLR4-dependent cell activation than wt LOS:MD-2 and partially antagonized the action of wt LOS:MD-2. These findings suggest that underacylated endotoxins produce decreased TLR4-dependent cell activation by altering the interaction of the endotoxin:MD-2 complex with TLR4 in a way that reduces receptor activation. Differences in potency among these endotoxin species is determined not by different aggregate properties, but by different properties of monomeric endotoxin:MD-2 complexes.     

Prostaglandin E2 and fever: a continuing debate

Prostaglandin (PG) E2 is a potent hyperthermic agent and has been assigned an intermediary function in the response of thermoregulatory neurons to pyrogens. Though attractive, this idea has been challenged on several grounds. The present study confirms that brain PGE2 synthesis increases during fever, the time course of the elevation according with a causative role of the compound. Our experimental data also argue against the involvement of a second cyclooxygenase product, specifically thromboxane (TX) A2, in the action of pyrogens. The sequence of events leading to PGE2 production and fever differs depending on the pyrogen, bacterial vs. leucocytic, and its route of administration. Blood-borne interleukin-1 (IL-1) acts on a discrete site in the central nervous system (CNS) which is tentatively identified with the organum vasculosum laminae terminalis (OVLT). The same site may also be the target for blood-borne endotoxin. In addition, endotoxin may promote PGE2 synthesis in the cerebral microvasculature. Both pyrogens, on the other hand, act diffusely throughout the CNS when given intrathecally. We conclude that PGE2 is well suited for an intermediary role in the genesis of fever and ascribe the reported inconsistencies to methodological factors.