Disposable TSM-biosensor based on viscosity changes of the contacting medium

The thickness shear mode (TSM)-sensor responds to changes of mechanical properties of the material contacting the surface of the sensor. One of the material properties is the viscosity of a liquid. Abiosensor based on the TSM-resonator for the detection of endotoxin has been developed. It exploits the viscosity–density change during the reaction of endotoxin with limulus amebocyte lysate (LAL). The effect of surface properties of the sensor has been investigated to achieve better output signals. It is shown that the sensor requires a hydrophilic surface to get a better coupling between the sensor and the LAL–endotoxin solution. The TSM biosensor is able to detect an endotoxin concentration as low as 100 fg/ml by using only 50-μl standard LAL solution. The disadvantages of reusable sensors, such as the contamination from previous measurement of endotoxin and the cost of the regeneration or reclining processes of the sensor, have been eliminated by using a cost effective disposable TSM-sensor.     

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.     

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.     

Monitoring blood coagulation with magnetoelastic sensors

The determination of blood coagulation time is an essential part of monitoring therapeutic anticoagulants. Standard methodologies for the measurement of blood clotting time require dedicated personnel and involve blood sampling procedures. A new method based on magnetoelastic sensors has been employed for the monitoring of blood coagulation. The ribbon-like magnetoelastic sensor oscillates at a fundamental frequency, which shifts linearly in response to applied mass loads or a fixed mass load of changing elasticity. The magnetoelastic sensors emit magnetic flux, which can be detected by a remotely located pick-up coil, so that no direct physical connections are required. During blood coagulation, the viscosity of blood changes due to the formation of a soft fibrin clot. In turn, this change in viscosity shifts the characteristic resonance frequency of the magnetoelastic sensor enabling real-time continuous monitoring of this biological event. By monitoring the signal output as a function of time, a distinct blood clotting profile can be seen. The relatively low cost of the magnetoelastic ribbons enables their use as disposable sensors. This, along with the reduced volume of blood required, make the magnetoelastic sensors well suited for at-home and point-of-care testing devices.     

人用狂犬病疫苗浓缩前后内毒素含量变化的研究

本文采用显色鲎试验法测定人用狂犬病疫苗浓缩前后半成品中内毒素含量。经鲎试验抑制增强实验确定人用狂犬病疫苗半成品对鲎试验法测定内毒素无干扰作用。样品经系列稀释,测定光吸收值（５４５ｎｍ）,然后将其对数回归直线与标准品的反应直线比较,结果表明,两者间有平行关系,经对四个生物制品研究所的样品进行测定,结果显示,人用狂犬病疫苗经超滤浓缩后内毒素含量有所增加,但在被检３１批样品中,除一所二批浓苗外,其余２９批均低于以往测得的最低家兔致热剂量（３．３２ＥＵ／ｍｌ／Ｋｇ）。     

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.     

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.     

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.     

Level of bacterial endotoxins in Haemophilus influenzae type b vaccines conjugated with toxoids (td, Di)

Quantitative evaluation of bacterial endotoxin was performed in the following vaccines: Act-Hib, Hiberix, Hib-Titer. The aim of this study was to assess the accuracy and precision of chromogenic LAL test with S-2423 substrate for this particular biopreparations and after that to determine the amounts of endotoxin as a factor of vaccine safety. Because of the lack of information concerning the presence of endotoxin in Act-Hib vaccine, we also tried to establish the limits for the presence of endotoxin in this type of vaccine. The estimated level of endotoxin was as follows: 110 EU/ml in Act-Hib, 1.64 EU/ml in Hiberix and 2.4 EU/ml in Hib-Titer. The results of this study showed that the amounts of endotoxin was dependent on the molecular size of polysaccharide PRP and on the presence of protein component. The limit of endotoxin presence in Act-Hib vaccine recommended by us is max. 150 EU/ml.     

Fabrication of sucrose biosensor based on single mode planar optical waveguide using co-immobilized plant invertase and GOD

In present studies, the new optical sensing platform based on optical planar waveguide (OPWG) for sucrose estimation was reported. An evanescent-wave biosensor was designed by using novel agarose–guar gum (AG) biopolymer composite sol–gel with entrapped enzymes (acid invertase (INV) and glucose oxidase (GOD)). Partially purified watermelon invertase isolated from Citrullus vulgaris fruit (specific activity 832 units mg⁻¹) in combination with GOD was physically entrapped in AG sol–gel and cladded on the surface of optical planar waveguide. Na⁺–K⁺ ion-exchanged glass optical waveguides were prepared and employed for the fabrication of sucrose biosensor. By addressing the enzyme modified waveguide structure with, the optogeometric properties of adsorbed enzyme layer (12 μm) at the sensor solid–liquid interface were studied. The OPWG sensor with short response time (110 s) was characterized using the 0.2 M acetate buffer, pH 5.5. The fabricated sucrose sensor showed concentration dependent linear response in the range 1 × 10⁻¹⁰ to 1 × 10⁻⁶ M of sucrose. Lower limit of detection of this novel AG–INV–GOD cladded OPWG sensor was found to be 2.5 × 10⁻¹¹ M sucrose, which indicates that the developed biosensor has higher sensitivity towards sucrose as compared to earlier reported sensors using various transducer systems. Biochips when stored at room temperature, showed high stability for 81 days with 80% retention of original sensitivity. These sucrose sensing biochips showed good operational efficiency for 10 cycles. The proper confinement of acid invertase and glucose oxidase in hydrogel composite was confirmed by scanning electron microscopy (SEM) images. The constructed OPWG sensor is versatile, easy to fabricate and can be used for sucrose measurements with very high sensitivity.     

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.     

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.     

Novel fluorescence method for real-time monitoring of nitric oxide dynamics in nanoscale concentration

A novel assay was developed for the measurement of nitric oxide. The proposed method is based on fluorescence, using a fluorophore-heme dual functionality probe (FHP). The heme group can serve as an effective NO-trap, due to its very fast reaction with NO and the high stability of the resulting complex. Since the heme is connected with a fluorophore as a part of the FHP dual-functionality probe, the heme can quench the fluorophore fluorescence, under certain conditions, by a singlet–singlet energy transfer mechanism.

The proposed method was tested using myoglobin covalently modified by a stilbene label. The change in emission intensity of the stilbene fragment, versus an increasing concentration of NO precursors, clearly demonstrated the spectral sensitivity required to monitor the formation of a heme–NO complex in a concentration range of 10 nM–2 μM. Furthermore, the new methodology for NO measurement was also found to be an effective assay using tissues from rabbit and porcine trachea epithelium. The measured NO flux (in an initial time interval) in tissue sample from rabbit trachea epithelia and porcine trachea epithelia is  7.9 × 10^− 12 mol/s × g and  3.0 × 10^− 12 mol/s × g respectively.     

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.     

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.     

A magnetoelastic bioaffinity-based sensor for avidin

A magnetoelastic bioaffinity sensor coupled with biocatalytic precipitation is described for avidin detection. The non-specific adsorption characteristics of streptavidin on different functionalized sensor surfaces are examined. It is found that a biotinylated poly(ethylene glycol) (PEG) interface can effectively block non-specific adsorption of proteins. Coupled with the PEG immobilized sensor surface, alkaline phosphatase (AP) labeled streptavidin is used to track specific binding on the sensor. This mass-change-based signal is amplified by the accumulation on the sensor of insoluble products of 5-bromo-4-chloro-3-indolyl phosphate catalyzed by AP. The resulting mass loading on the sensor surface in turn shifts the resonance frequency of the magnetoelastic sensors, with an avidin detection limit of approximately 200 ng/ml.     

A performance comparison of choline biosensors: anodic or cathodic detections of H2O2 generated by enzyme immobilized on a conducting polymer

Amperometric choline biosensors were fabricated by the covalent immobilization of an enzyme of choline oxidase (ChO) and a bi-enzyme of ChO/horseradish peroxidase (ChO/HRP) onto poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (poly-TTCA) modified electrodes (CPMEs). A sensor modified with ChO utilized the oxidation process of enzymatically generated H₂O₂ in a choline solution at +0.6 V. The other one modified with ChO/HRP utilized the reduction process of H₂O₂ in a choline solution at −0.2 V. Experimental parameters affecting the sensitivity of sensors, such as pH, applied potential, and temperature were optimized. A performance comparison of two sensors showed that one based on ChO/HRP/CPME had a linear range from 1.0×10⁻⁶ to 8.0×10⁻⁵ M and the other based on ChO/CPME from 1.0×10⁻⁶ to 5.0×10⁻⁵ M. The detection limits for choline employing ChO/HRP/CPME and ChO/CPME were determined to be about 1.0×10⁻⁷ and 4.0×10⁻⁷ M, respectively. The response time of sensors was less than 5 s. Sensors showed good selectivity to interfering species. The long-term storage stability of the sensor based on ChO/HRP/CPME was longer than that based on ChO/CPME.     

An in vitro method for evaluating endotoxic activity using prostaglandin E2 induction in bovine peripheral blood

Severe side effects of veterinary vaccines, in particular Histophilus somni-containing vaccines for cows, have frequently been reported in Japan. These side effects are probably caused by endotoxins. Contamination levels of endotoxins could be monitored using the Limulus amebocyte lysate (LAL) test; however, the LAL test is not completely adequate for evaluation of in vivo endotoxic activities. In this study, we established a method for evaluating endotoxic activities using prostaglandin E₂ (PGE₂) induction in bovine peripheral blood. Blood and standard endotoxin, derived from Escherichia coli, were mixed and incubated. The concentration of induced PGE₂ in the culture supernatant reached a maximum after 24-h incubation. A linear dose-response curve was observed for PGE₂ concentration and the logarithmic transformed standard endotoxin concentration (5–5000 ng/ml). The endotoxic activity of H. somni in cows was the highest among those of several tested endotoxins. However, the LAL activities of H. somni were not as high as those of the other tested endotoxins. These results may provide a reason for the many report of side effects of H. somni-containing vaccines. The PGE₂ detection assay described here could be a valuable method for evaluating the endotoxic activities of vaccines in cows.     

Feasibility of on-chip detection of endotoxin by LAL test

The LAL (Limulus amebocyte lysate) test for the detection and quantification of endotoxin is based on the gelation reaction between endotoxin and LAL from a blood extract ofLimulus polyphemus. The test is labor intensive, requiring dedicated personnel, a relatively long reaction time (approximately 1 h), relatively large volumes of samples and reagents and the detection of the end-point is rather subjective. To solve these problems, a miniaturized LOC (labon-a-chip) prototype, 62 mm (L)×18 mm (W), was fabricated using PDMS (polydimethylsiloxane) bonded to glass. Using this prototype, in which 2 mm (W)×44.3 mm (L)×100 μm(D) microfluidic channel was constructed, turbidometric and chromogenic assay detection methods were compared, and the chromogenic method was found the most suitable for a small volume assay. In this assay, the kinetic-point method was more accurate than the end-point method. The PDMS chip chickness was found to be minimized to around 2 mm to allow sufficient light transmittance, which necessitated the use of a glass slide bonding for chip rigidity. Due to this miniaturization, the test time was reduced from 1 h to less than 10 min, and the sample volume could be reduced from 100 toca. 4.4 μL. In summation, this study suggested that the LOC using the LAL test principle could be an alternative as a semi-automated and reliable method for the detection of endotoxin.