Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product.

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Limulus amoebocyte lysate assay for detection and quantitation of endotoxin in a small-volume parenteral product

A Limulus amoebocyte lysate gel-clotting method for the determination of endotoxin in a small-volume parenteral product has been described. Sample dilution with 0.1 M potassium phosphate monobasic buffer (pH 8.0) effectively eliminated assay interference, whereas dilution with water did not. The threshold pyrogenic dose for Escherichia coli EC-2 and O127:B8 endotoxins was determined to be 1.0 ng of endotoxin per kg of body weight. Not more than 1.0 ng of endotoxin (the threshold pyrogenic dose) per the highest recommended human dose or the USP pyrogen test dose per kg of body weight, whichever dose is more stringent, is a logical limit for the quantity of bacterial endotoxin in small-volume parenteral products. Excellent correlation was attained when this criterion was used to compare the Limulus amoebocyte lysate assay with the USP pyrogen test.     

Evaluation of the bacterial endotoxin test for quantification of endotoxin contamination of porcine vaccines.

We investigated the application of the bacterial endotoxin test for the quantification of the endotoxin contamination of various commercial porcine vaccines. In endotoxin-spiked samples, Freund's complete adjuvant and aluminum hydroxide gel adjuvant failed to interfere with the results of the endotoxin test, and both recovery ratios were within the permissible range mentioned in the Japanese Pharmacopoeia. At the various dilutions tested, none of the adjuvants in commercial porcine vaccines caused noteworthy interference in the test. In addition, none of the 39 samples of porcine vaccines approved in Japan induced an interfering effect in the endotoxin test. Our findings suggest that the bacterial endotoxin test using endotoxin-specific Limulus amoebocyte lysate (LAL) can detect endotoxin contamination in commercial porcine vaccines containing either oil or aluminum adjuvants.     

Development of an endotoxin-specific Limulus amebocyte lysate test blocking beta-glucan-mediated pathway by carboxymethylated curdlan and its application

We developed a simple new endotoxin-specific assay method that uses Limulus amebocyte lysate (LAL) containing a sufficient amount of a water-soluble (1----3)-beta-D-glucan derivative as a blocker of the (1----3)-beta-D-glucan-mediated coagulation pathway. The addition of 0.1 mg/ml or more of carboxymethylated (1----3)-beta-D-glucan completely blocked the activation of LAL by (1----3)-beta-D-glucan itself. The assay of endotoxin was unaffected by the presence of 1 mg/ml carboxymethylated (1----3)-beta-D-glucan. Spiked endotoxin was recovered well from beta-glucans by the turbidimetric kinetic method with LAL containing 1 mg/ml of carboxymethylated (1----3)-beta-D-glucan. Besides, this new LAL formulation was applied for an endotoxin-specific assay by the conventional gel-clot method or the chromogenic method. Gram-negative bacteria were specifically detected by the turbidimetric kinetic method with the LAL formulation. This LAL formulation may be used for an endotoxin-specific assay not only in pharmacology but also in clinical microbiology.     

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study.

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.     

Comparison of several control standard endotoxins to the National Reference Standard Endotoxin--an HIMA collaborative study

A collaborative study, initiated under the auspices of the Health Industry Manufacturers Association (HIMA), was designed to establish the relationship of Escherichia coli O55:B5 endotoxin (the control standard endotoxin of HIMA and the Food and Drug Administration's Office of Medical Devices) to the U.S. National Reference Standard Endotoxin and to two internationally used control standard endotoxins. By using two Limulus amoebocyte lysate test systems, it was established that the E. coli O55:B5 endotoxin lot originally used by HIMA and the Office of Medical Devices to establish Limulus amoebocyte lysate release test criteria for pyrogen testing of medical devices contains approximately 4.5 endotoxin units (EU) per ng. Thus, the 1.0-ng/kg endotoxin dose limit currently established for medical devices is approximately the same as the 5.0-EU/kg endotoxin limit (on an activity basis) established by several other Food and Drug Administration agencies for human and animal parenteral drugs and biological products.     

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.     

Evaluation of the applicability of the bacterial endotoxin test to antibiotic products.

Fundamental conditions for applying the bacterial endotoxin test to antibiotic products were investigated so as not to affect the level of regulation by the rabbit pyrogen test. According to accuracy evaluation of test methods, the kinetic-turbidimetric and kinetic-colourimetric assays were shown to allow more accurate measurement and, therefore, more sensitive detection of interference to the bacterial endotoxin test than the gel-clot method. In total, 102 antibiotic products were evaluated on their interfering effect to show that the antibiotics could be categorized into three groups depending on intensity of the interference. Although the test was shown to be applicable even to the group showing the strongest interference, it was assumed to be crucial to use appropriate reagents and an accurate test method for avoiding approval of a pyrogenic product. Accordingly, lists of antibiotics are presented to provide limits of concentration for eliminating interference and endotoxin limits for approval to facilitate effective bacterial endotoxin tests.     

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。     

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。     

Relative potencies of four reference endotoxin standards as measured by the Limulus amoebocyte lysate and USP rabbit pyrogen tests.

Four commonly used reference endotoxin standards, Escherichia coli O113:H10:K0, E. coli O55:B5, Salmonella abortusequi, and Shigella dysenteriae were compared by the USP rabbit pyrogen and the Limulus amoebocyte lysate tests. By the rabbit pyrogen test, S. abortus equi was identified as the most potent endotoxin, followed closely by E. coli O113:H10:K0 and E. coli O55:B5.     

Chromatographic removal of endotoxin from protein solutions by polymer particles

Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products. Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions. Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective. In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents. We also provide the design of novel endotoxin-specific polymer adsorbents.     

Residual Endotoxin Contaminations in Recombinant Proteins Are Sufficient to Activate Human CD1c+ Dendritic Cells

Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU). When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c⁺ dendritic cells (DCs) with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002–2 ng/ml). We show that CD1c⁺ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c⁺ DCs was closely correlated with high CD14 expression levels observed in CD1c⁺ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.     

Characterization of Limulus amoebocyte lysate-reactive material from hollow-fiber dialyzers.

Hollow-fiber hemodialyzers containing cellulose-based membranes have been shown to produce positive results with the Limulus amoebocyte lysate test. This study was undertaken to determine whether endotoxin was causing the reaction. Rinses from 45 parallel-plate and hollow-fiber dialyzers from eight different manufacturers were tested before and after treatment with cellulase, using three lysates and four Limulus amoebocyte lysate methods. In addition, four in vitro cellular methods--human leukocytic pyrogen, lymphocytic activating factor, peritoneal macrophage, and arginase release--were used to evaluate endotoxin activity. The substance causing the reaction was identified by chromatographic methods. Results indicate that the Limulus amoebocyte lysate reactive material is cellulose derived and not pyrogenic.     

Construction of a sensitive pyrogen-testing cell model by site-specific knock-in of multiple genes

A pyrogen test is crucial for evaluating the safety of drugs and medical equipment, especially those involved in injections. As existing pyrogen tests, including the rabbit pyrogen test, the limulus amoebocyte lysate (LAL) test and the monocyte activation test have limitations, development of new models for pyrogen testing is necessary. Here we develop a sensitive cell model for pyrogen test based on the lipopolysaccharides (LPS) signal pathway. TLR4, MD2, and CD14 play key roles in the LPS-mediated pyrogen reaction. We established a new TLR4/MD2/CD14-specific overexpressing knock-in cell model using the CRISPR/CAS9 technology and homologous recombination to detect LPS. Stimulation of our TLR4/CD14/MD2 knock-in cell line model with LPS leads to the release of the cytokines IL-6 and TNF-alpha, with a detection limit of 0.005 EU/ml, which is greatly lower than the lower limit of 0.015 EU/ml detected by the Tachypleus amebocyte lysate (TAL) assay.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

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.     

Sodium-Dependent Growth of Azotobacter chroococcum

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.     

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     

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.