A novel endotoxin-specific assay by turbidimetry with Limulus amoebocyte lysate containing beta-glucan

The gelation of standard Limulus amoebocyte lysate (LAL) is triggered by the addition of a small amount of beta-glucan (1-1000 ng/ml plasma), but in the presence of an excessive amount of beta-glucan (1 mg/ml plasma), the gelation becomes insensitive to beta-glucan. Utilizing this property, a method to determine quantitatively the amount of endotoxin circulating in humans was developed. When a modified LAL, or LAL-ES, which contains an excessive amount of CM-curdlan as beta-glucan, was used for the assay, a linear relation in the logarithmic scales was obtained between the gelation time measured by the turbidimetry (min) and the concentration of endotoxin. This relation was not affected by a considerable amount of beta-glucan (100 ng/ml). The sensitivity of the endotoxin assay was estimated to be as low as 3 pg/ml. The following aspects of the method were found by clinical application to normal and febrile subjects. (1) Using both LAL and LAL-ES, it was possible to distinguish the effect of endotoxin from that of beta-glucan in plasma, i.e., bacterial sepsis from fungal sepsis. (2) The amount of circulating endotoxin determined by the present method showed good correlation to those obtained by chromogenic assay using modified LAL devoid of Factor G which could be activated by beta-glucan.     

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.     

Automation of chromogenic substrate Limulus amebocyte lysate assay method for endotoxin by robotic system.

The chromogenic substrate Limulus amebocyte lysate (LAL) assay method for the detection of endotoxin was automated by a Zymate robotic system. The software developed enables the robot to automatically dilute a stock reference endotoxin standard (20,000 endotoxin units per ml) for the construction of a five-point standard curve, make sample dilutions to the proper testing concentration, and perform chromogenic substrate LAL assays in duplicate. The linearity of the standard curve and the endotoxin concentration in each sample are calculated and results are printed automatically. In 48 min the automated system assays three samples and a reference standard in duplicate along with a water blank. Sensitivity of the assay is a function of incubation time. The assay is linear (r greater than 0.99) in the region of 0 to 1.0 endotoxin units per ml or 0 to 0.2 endotoxin units per ml with incubation times of 10 or 16 min, respectively. The method can be made very sensitive, detecting as low as 0.003 endotoxin units per ml with 30 min of incubation. The precision of the assay method, determined by assaying an endotoxin reference solution eight times, is ca. 6%. The LAL reagent designed for gel-clot assay was modified for the chromogenic substrate assay. We describe the optimum conditions for the performance of the chromogenic substrate LAL assay and stability of the LAL reagent.     

Selective assay for endotoxin using poly(ε-lysine)-immobilized Cellufine and Limulus amoebocyte lysate (LAL)

We developed a selective endotoxin (lipopolysaccharide; LPS) assay using poly(ε-lysine)-immobilized cellulose beads (PL-Cellufine) and Limulus amoebocyte lysate (LAL). First, LPS was selectively adsorbed on the beads in a solution containing various LAL-inhibiting or LAL-enhancing compounds (e.g., amino acids, enzymes) and the LPS adsorbed on the beads was separated from the compounds by centrifugation. Second, the LPS adsorbed on the beads directly reacted with the LAL reagent, and the LPS concentration was determined by a turbidimetric time assay. The accuracy of the adsorption method with PL-Cellufine was high compared with that of a common solution method. Apparent recovery of LPS from compound solution was 88-120%.     

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.     

Development of an electrochemical Limulus amebocyte lysate assay technique for portable and highly sensitive endotoxin sensor

Here, we report the development of an electrochemical detection method for endotoxin based on the Limulus amebocyte lysate (LAL) assay. A mixture of LAL reagent and endotoxin sample solution was incubated for 1 h. The endotoxin activated a cascade reaction of zymogens contained in the LAL to generate p-nitroaniline (pNA) which was then electrochemically detected by differential pulse voltammetry (DPV). The generated pNA gave a clear peak at -0.75 V vs. silver/silver chloride (Ag/AgCl), which increased with the concentration of endotoxin in the LAL assay solution. This DPV detection was performed using an electrode chip device fabricated from a diamond-like carbon-coated glass substrate. This chip device could detect as low as 10 endotoxin units l(-1) at room temperature within 1 h. This novel electrochemical method for the detection of endotoxin appears promising for the development of compact, low-cost and easy-to-use sensors for on-site monitoring of potentially contaminated medical supplies, including dialysis fluid, transplanted tissue and culture medium for assisted reproduction.     

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.     

A rapid highly-sensitive endotoxin detection system

This paper presents a rapid, highly-sensitive, and low-cost method of endotoxin quantification based on the use of stress-responsive magnetoelastic sensors, that monitor the gel formation (viscosity change) of the Limulus Amoebocyte Lysate (LAL) assay in response to endotoxin. Ribbon-like magnetoelastic sensors, 12.7 mm × 6 mm × 28 μm, were immersed in a LAL assay after mixing with test samples of variable endotoxin concentration, and the decrease in resonance amplitude of the sensor was recorded as a function of time. Experimental results show excellent correlation between endotoxin concentration and the maximum clot rate, determined by taking the minimum point of the first derivative of the amplitude–time curve, as well as the clotting-time, defined as the time that corresponds to the maximum clot rate. Using a LAL gel–clot assay with a sensitivity of 0.06 EU/ml (EU: endotoxin unit), the magnetoelastic sensor based technology can detect the presence of endotoxin at 0.0105 EU/ml in test requiring approximately 20 min. Unlike optical methods used for determining endotoxin concentration, the color of the test solution does not impact the magnetoelastic sensor measurement. Due to the small size of the sensor reader electronics and low cost, the magnetoelastic sensor based endotoxin detection system is ideally suited for wide-spread use in endotoxin screening for sepsis prevention.     

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.     

Synthesis and antitumour activity of sulfoalkyl derivatives of curdlan and lichenan.

2-Sulfoethyl, 3-sulfopropyl, and 4-sulfobutyl derivatives of the (1----3)-beta-D-glucan curdlan and the (1----3/1----4)-beta-D-glucan lichenan have been synthesised. The substituents are located mainly at positions 6. The curdlan derivatives strongly inhibited the growth of the Sarcoma 180 tumour, whereas the lichenan derivatives were inactive, indicating that a (1----3)-linked beta-D-glucan backbone is essential for activity.     

18-氨基酸注射液的动态浊度法检测研究

建立 18 -氨基酸注射液细菌内毒素定量测定方法 ,控制药品质量。采用动态浊度法 ,对 18-氨基酸注射稀释液进行干扰预实验、干扰实验定量检测。结果 18-氨基酸注射液在 8倍稀释时无干扰作用。因此用动态浊度法定量检查 18-氨基酸注射液细菌内毒素的含量 ,结果准确 ,在实际应用中完全可行。     

Early detection of the Limulus amebocyte lysate reaction evoked by endotoxins

The gelation of Limulus amebocyte lysate (LAL) evoked by bacterial endotoxins can be detected earlier than with usual methods by using laser scattering photometry to recognize the formation of small particles of clotted enzyme produced when the reaction mixture is agitated. The appearance of these small particles means that the influence of endotoxins has stimulated activation of the clotting enzyme across the LAL cascade, and the timing of their appearance is related to endotoxin concentration. This new method can be used for quick and sensitive endotoxin assay. The average endotoxin level of healthy volunteers was assayed to be 0.0738 pg/ml [0.0312-0.3445 pg/ml] (n = 11) within 70 min from the start of the assay.     

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.     

The yeast KRE5 gene encodes a probable endoplasmic reticulum protein required for (1----6)-beta-D-glucan synthesis and normal cell growth.

Yeast kre mutants define a pathway of cell wall (1----6)-beta-D-glucan synthesis, and mutants in genes KRE5 and KRE6 appear to interact early in such a pathway. We have cloned KRE5, and the sequence predicts the product to be a large, hydrophilic, secretory glycoprotein which contains the COOH-terminal endoplasmic reticulum retention signal, HDEL. Deletion of the KRE5 gene resulted in cells with aberrant morphology and extremely compromised growth. Suppressors to the KRE5 deletions arose at a frequency of 1 in 10(7) to 1 in 10(8) and permitted an analysis of deletions which were found to contain no alkali-insoluble (1----6)-beta-D-glucan. These results indicate a role for (1----6)-beta-D-glucan in normal cell growth and suggest a model for sequential assembly of (1----6)-beta-D-glucan in the yeast secretory pathway.     

An immunoassay for the measurement of (1-->3)-beta-D-glucans in the indoor environment

An inhibition enzyme immunoassay was developed for quantitation of (1-->3)-beta-D-glucans in the indoor environment. Immunospecific rabbit antibodies were produced by immunization with bovine serum albuminconjugated laminarin.The laminarin calibration curve ranged from 40 to 3000 ng/ml.Another (1-->3)-beta-D-glucan (curdlan) showed a similar inhibition curve, but was less reactive on a weight basis. Pustulan, presumed to be (1-->3)-beta-D-glucan, also showed immunoreactivity in the assay. Control experiments indicated that this was due to (1-->3)-beta-D-glucan structures. Other non-(1-->3)-beta-D-glucan polysaccharides did not react. (1-->3)-beta-Dglucan was detectable in dust from a variety of occupational and environmental settings. We conclude that the new assay offers a useful method for indoor (1-->3)-beta-Dglucan exposure assessment.     

Inhalation of (1-->3)-beta-D-glucan causes airway eosinophilia

BACKGROUND: Moulds are present in a variety of environments and aerosols of fungal spores are generated when mouldy materials are handled. Molds contain (1-->3)-beta-D-glucan, a polyglucose which is present in the cell wall of fungi, certain bacteria and plants. AIM: This study was undertaken to investigate the cellular inflammatory response in the lung after inhalation of (1-->3)-beta-D-glucan and bacterial endotoxin. METHODS: Guinea pigs were exposed daily to an aerosol of pure (1-->3)-beta-D-glucan and pure endotoxin for five weeks. Lung lavage and lung interstitial cell preparations were done and the inflammatory cells counted. Histological sections were prepared from the trachea. RESULTS: There was an increase in eosinophil numbers in lung lavage, lung interstitium, and the airway epithelium of animals exposed to (1-->3)-beta-D-glucan. In animals simultaneously exposed to endotoxin, there was no increase in eosinophils. In the lung interstitium, (1-->3)-beta-D-glucan exposure caused an increase in lymphocytes, which was not found after endotoxin exposure. Endotoxin exposure caused an increase in neutrophils and macrophages in lung lavage, which was not found after (1-->3)-beta-D-glucan exposure. CONCLUSIONS: The results support previous findings that (1-->3)-beta-D-glucan causes a different response in the airways as compared to endotoxin. Endotoxin modulated the increase in eosinophils caused by (1-->3)-beta-D-glucan exposure, suggesting a complex interaction between the microbial cell wall components.     

The Release and Detection of Endotoxin from Liposomes

Incorporation of lipopolysaccharide (LPS) into liposomes dramatically reduces its ability to coagulateLimulusamebocyte lysate (LAL). The coagulation of LAL is commonly used to signal the presence of endotoxinin vitro.This study demonstrates a simple method to release masked endotoxin from liposomal dispersions using moderate amounts of detergent to form mixed micelles containing lipid, detergent, and LPS. Several parameters were found to affect the degree of liposome solubilization and/or the sensitivity of the LAL assay. These included detergent type and concentration, temperature for solubilization, lipid composition, liposome morphology, and time for test incubation. The nonionic detergent polyoxyethylene 10 lauryl ether (C₁₂E₁₀) proved to be unique in its ability to solubilize liposomes and minimally interfere with endotoxin detection. The LAL endotoxin detection limit for samples dispersed in C₁₂E₁₀varied with the phospholipid component; the sensitivity decreased in the order DSPC > DPPC = EPC DMPC. Cholesterol lowered the solubility limit of the liposomes, but did not appear to affect the LAL assay sensitivity once the liposomes were completely solubilized. The presence of negatively charged phospholipids, DSPG and Pops, also lowered the solubility limit. Pops, but not DSPG, at 10 mol% further decreased the LAL endotoxin detection limit. This detergent-solubilization method should be useful in liposomal LPS immunological studies or in other situations where accurate determination of endotoxin concentration is important.     

Microbial cell wall product contamination of bedding may induce pulmonary inflammation in rats

To test the hypothesis that airborne microbial cell wall components could induce an inflammatory response in the lungs, measurements were made of the amounts of bacterial endotoxin and (1-->3)-beta-D-glucan in laboratory animal bedding materials. Groups of rats were exposed by inhalation to airborne endotoxin, (1-->3)-beta-D-glucan or a combination of the two for 5 weeks. The results demonstrated that measurable amounts of endotoxin and (1-->3)-beta-D-glucan could be detected in the different bedding materials. In contrast to animals at delivery, those kept on bedding for 5 weeks showed moderate inflammatory reactions in the lung. These were most pronounced among animals exposed to endotoxin and (1-->3)-beta-D-glucan. The results suggest that further studies need to be undertaken to elucidate the role of microbial cell wall products in the development of inflammatory lung responses among research animals.     

A limulus amoebocyte lysate activating activity (LAL activity) that lacks biological activities of endotoxin found in biological products

Pyrogenic substances in influenza HA (IHA) vaccine have been controlled by the pyrogen test or the mouse body weight decreasing toxicity (BWD) test. We examined the possibility of replacing the animal tests with the endotoxin test Commercial IHA vaccines were found to show considerable levels of LAL activity ranging from 0.2 to 160 EU/ml. However, a batch of the vaccine having even 100 EU/ml of LAL activity showed neither pyrogenicity in rabbits nor tumor necrosis factor alpha (TNF-alpha) induction in RAW264.7 cells. The LAL activity of IHA vaccine was abolished by a monoclonal antibody that recognizes LPS-binding epitope of LAL factor C. The activity of IHA vaccine showed different physicochemical properties from those of LAL activity of endotoxin. LAL activity of endotoxin is known to be sensitive to polymyxin B treatment and was found to be resistant to polyoxyethylene 10 cetyl ether (Brij56) treatment. On the contrary, the LAL activity of IRA vaccine was shown to be resistant to polymyxin B but sensitive to Brij56 treatment. The difference in sensitivity of the two LAL activities to polymyxin B and Brij56 might suggest the possibility of their discriminative measurements.     

Applicability of bacterial endotoxins test to various blood products by the use of endotoxin-specific lysates

Endotoxin contamination is a serious threat to the safety of parenteral drugs, and the rabbit pyrogen test has played a crucial role in controlling this contamination. Although the highly sensitive endotoxin test has replaced the pyrogen test for various pharmaceuticals, the pyrogen test is still implemented as the control test for most blood products in Japan. We examined the applicability of the endotoxin test to blood products for reliable detection and quantification of endotoxin. Nineteen types of blood products were tested for interfering factors based on spike/recovery of endotoxin by using 2 types of endotoxin-specific lysate reagents for photometric techniques. Interfering effects on the endotoxin test by the products could be eliminated by diluting from 1/2 to 1/16, with the exception of antithrombin III. However, conventional lysate reagents that also react with non-pyrogenic substances, such as (1–3)-β-d-glucan, produced results that were not relevant to endotoxin content or pyrogenicity. Our results showed that the endotoxin test would be applicable to most blood products if used with appropriate endotoxin-specific lysate reagents.