Standard Endotoxin Research Articles

Uniform Suspension of Heat-Killed Staphylococcus aureus for a Positive Control Used in the Monocyte-Activation Test.

Pyrogens, classified as bacterial endotoxins and non-endotoxin pyrogens (NEPs), induce fever or shock when released into the bloodstream or spinal fluid. Recently, a monocyte-activation test (MAT) involving human cell culture has been developed to detect pyrogens in injectable products. To evaluate the sensitivity of MAT, a reference standard endotoxin was used as a positive control; however, the reactivity differed between the endotoxins and NEPs, necessitating positive controls for NEPs. This study aimed to explore a preparation method for heat-killed Staphylococcus aureus (HKSA) as a positive control for NEPs in MAT. Because S. aureus forms grape-like clusters, nine types of glass filters with pore sizes of 0.5-2.7 µm were evaluated to obtain a uniform bacterial suspension. The suspension was then heat-treated to kill the bacteria, resulting in HKSA samples. Serial dilutions of HKSA were tested by MAT using peripheral blood mononuclear cells. The interleukin-6 concentrations in the culture supernatant were measured by enzyme-linked immuno-sorbent assay to assess pyrogenic activities of HKSA. The pore sizes of the glass filters affected the uniformity of HKSA, and GF/C filter was selected for HKSA preparation. Repeated filtration improved uniformity, and a uniform suspension of HKSA was obtained through double filtration using a GF/C filter. Despite the decrease in HKSA activity as filtration frequency increased, the detection limit remained consistently unchanged. This suggests that repeated filtration can adjust the activity of HKSA to a baseline level and that a uniform suspension of HKSA exhibiting low variation is suitable as a positive control in MAT.

It is time to reconsider the use of naturally-occurring endotoxins in endotoxin recovery studies: Part 2 of a BioPhorum harmonized endotoxin recovery study

The evaluation of Naturally Occurring Endotoxins (NOEs) for Low Endotoxin Recovery (LER) studies has been a topic in the industry and regulatory agencies have been hesitant to endorse NOE use in LER studies over purified Lipopolysaccharide (LPS) standards such as Control Standard Endotoxin (CSE) or Reference Standard Endotoxin (RSE). In a recent study involving 11 BioPhorum member companies across 13 sites, NOEs prepared in high and low nutrient conditions were evaluated in two common monoclonal antibody buffer formulations: 10 mM Sodium Citrate, 0.05 % Polysorbate 80, pH 6.0 and 20 mM Histidine, 0.05 % Polysorbate 80, pH 6.0. 12 g-negative bacterial isolates were used to prepare NOE analytes, which were spiked into the formulation buffers. Additionally, the NOEs were spiked into Limulus Amebocyte Lysate (LAL) reagent water as controls and purified LPS into the citrate/polysorbate buffer as the LER control. Results showed the average of three runs per organism was >50 % recovery, at the conclusion of the 7-day period, regardless of nutrient culture preparation conditions. Furthermore, purified LPS controls became undetectable (<50 % recovery) in the citrate/polysorbate buffer, highlighting the presence of LER. These findings highlight the potential value of using NOEs from relevant manufacturing facilities to assess overall risk when purified LPS recovery is insufficient.

A Study on the Application of Recombinant Factor C (rFC) Assay Using Biopharmaceuticals.

Gram-negative bacterial endotoxins can cause pathophysiological effects such as high fever when introduced into the bloodstream. Therefore, endotoxin testing is necessary when producing injectable pharmaceuticals. The pharmaceutical industry has widely used Limulus amebocyte lysate (LAL) to certify product quality. However, ethical concerns have been raised and the increasing scarcity of Limulus polyphemus necessitates the development of novel testing techniques. Recombinant factor C (rFC) was developed using genetic engineering techniques. The aim of this study was to investigate the validity of rFC testing and compare it with the LAL method. The specificity, linearity, accuracy, precision, and robustness of the rFC assay were evaluated. After validation, the rFC assay was found to be suitable for endotoxin detection. We compared the accuracy of the rFC and LAL assays using reference standard endotoxin. The rFC assay was as accurate as the LAL assay. We also compared the two assays using biopharmaceuticals. Greater interference occurred in some samples when the rFC assay was used than when the LAL assay was used. However, the rFC assay overcame the interference when the samples were diluted. Overall, we suggest that rFC can be applied to test biopharmaceuticals.

Characterization of Trypsin-like Serine Proteases from Tachypleus tridentatus Amebocyte Lysate and its Potential for Endotoxin Detection

Background: Serine Proteases in the Tachypleus Amoebocyte Lysate (TAL) from horseshoe crab, Tachpleus tridentatus have many applications in biomedical industries. The sensitivity of these enzymes is a crucial factor for such usages. Until today, the catalytic behavioural and biochemical stability of these enzymes has not been properly addressed. This report aimed to characterize the physicochemical properties of serine proteases to determine their kinetic reactions and to determine the potential utilization of the enzymes for endotoxin detection. Methods: The purification process of protease from crude TAL was performed using a two-phase filtration unit by affinity and gel columns and the molecular weights of the enzyme fraction from crude, affinity and gel filtration columns were determined. The Azocasein, Aldolase and transaldolase assays were used for the determination of the enzyme Catalysis. The chromogenic assay was carried out to determine the bacterial endotoxin effects using a standard endotoxin from strain Escherichia coli 0113: H10 as a positive control. The absorbance was measured at 405 nm. The enzyme activity was determined using the constructed p-nitroanilide standard curve. Result: A 72 kDa trypsin-like enzyme was purified at fraction-14 (F14) gel filtration and identified as transaldolase. The purified-F14 hydrolysed trypsin substrate, Ná-Benzoyl-L-Arginine-p-nitroanilide at higher activity than chymotrypsin. In conclusion, the purified enzyme showed a great potential for enhancing the TAL sensitivity.

Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery.

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

Depyrogenation using Plasmas: A Novel Approach for Endotoxin Deactivation Using a Dielectric Barrier Discharge at Atmospheric Pressure.

Developing a low-cost depyrogenation process is vital in extending medical applicability of polymers that can be used in medicine. We present an overview of the plasma-based depyrogenation literature and address the need to develop a non-thermal plasma-based depyrogenation process for delicate materials such as chitosan. We present a low-cost plasma apparatus to treat chitosan powder in hermetically sealed bags. We decouple the experiments into two; depyrogenation experiments for dried standard endotoxin on glass slides, and chitosan modifications analysis through FTIR spectroscopy. We demonstrate depyrogenation efficacy with up to a 4-log reduction in endotoxin levels and discuss minor changes observed in plasma-treated chitosan.

Low endotoxin recovery and its impact on endotoxin detection.

Endotoxin exists on the outer membrane of Gram-negative bacteria and poses risks to human health by triggering a series of immune responses. Therefore, its accurate detection is essential. The Limulus amoebocyte lysate (LAL) test is the most pharmacopeia-recognized and popular technique for endotoxin detection. Despite its wide industry adoption, the low endotoxin recovery (LER) phenomenon can compromise the LAL test's reliability. This review summarizes the possible reasons attributing to the LER phenomenon from three different perspectives: the endotoxin standards used in hold time study, protein active pharmaceutical ingredients, and excipients. Potential mechanisms and strategies to mitigate the LER phenomenon are also discussed as presented by different research groups.

Efficacy of a Fixed-Dose Combination of Ibuprofen and Acetaminophen Compared With Individual Monocomponents in Adult Male Subjects With Endotoxin-Induced Fever: A Randomized Controlled Trial

PurposeThis study evaluated antipyretic efficacy and onset of a novel fixed-dose combination (FDC) of ibuprofen (IBU; 250 mg) and acetaminophen (APAP; 500 mg) compared with placebo and IBU or APAP monocomponents. MetThis single-center, randomized, double-blind, placebo-controlled, full-factorial study was conducted in healthy males aged 18 to 55 years with pyrexia induced by intravenous administration of reference standard endotoxin (RSE). After attainment of an oral temperature ≥38.1°C, subjects were randomized 3:3:3:1 to a double-blind single oral dose of FDC IBU/APAP 250 mg/500 mg, APAP 500 mg, IBU 250 mg, or placebo. Oral temperature was measured every 10 minutes for 2 hours, then every 30 minutes until 8 hours postdose. Time-weighted sum of temperature differences from baseline to 8 hours (WSTD0–8) after study medication administration was the primary efficacy end point. Secondary end points included WSTD scores from 0 to 2 hours, 0 to 4 hours, 0 to 6 hours, and 6 to 8 hours; time to return to “normal” temperature; time to rescue medication use; and global drug evaluation. Safety was assessed via adverse events (AEs). FindingsTwo hundred ninety subjects were randomized; 273 were included in the primary efficacy analysis. WSTD0–8 was significantly better for FDC IBU/APAP 250 mg/500 mg (P = 0.002), IBU 250 mg (P = 0.030), and APAP 500 mg (P = 0.023) versus placebo; there were no significant differences between active treatments. For WSTD0–2, only the FDC was statistically significant versus placebo (P = 0.004). All active treatments were significantly better (P < 0.05) for WSTD0–4 and WSTD0–6 versus placebo; there were no differences in WSTD6–8 between cohorts. Temperature returned to normal during the 8-hour treatment period in ∼50% of subjects in each cohort. Only 1 subject (IBU cohort) took rescue medication. Post hoc analyses at early time points revealed significant treatment differences favoring FDC versus placebo and IBU for the WSTD from baseline during the 50- to 110-minute posttreatment window; for WSTD from baseline during the 80- to 110-minute posttreatment window, FDC provided significant treatment differences versus placebo and both monocomponents. Overall, 223 (76.9%) of 290 subjects experienced AEs related to RSE; only 2 subjects experienced treatment-related AEs (FDC, rash; placebo, ear pain). ImplicationsAlthough the primary end point was not met, these results suggest that FDC IBU/APAP 250 mg/500 mg provides effective antipyresis with a faster onset versus equal doses of IBU and APAP alone. ClinicalTrials.gov identifier: NCT02761980.

Development of innovative technology for inactivation of endotoxins at low temperature condition utilizing gas phase sterilization system

Background & Aim With the rapid development of gene and cell therapy, it is increasingly important to address the continuing risk concerns of endotoxins involving in the manufacturing and quality control of medical products and devices. Although dry-heat sterilization is generally considered as the preferred method for rendering heat-stable materials free of endotoxin, there are limitations to the effective approaches available to inactivate bacterial endotoxin, especially at low temperature. We have developed innovative technology as gas phase sterilization system based on a catalytic reaction mechanism by use of methanol to generate mixed biogas, exhibiting remarkable performance of nucleic acid decomposition as well as sterilization. Here we will present our recent challenge of findings of additional remarkable function of this system exhibiting inactivation of bacterial endotoxin at even low temperature around room temperature. Methods, Results & Conclusion Standard endotoxin indicators 2,000 EU/vial (2K-ET) (Charles River Laboratores, CRL) were used for the experiments, and endotoxin concentrations were determined by Endosafe®-PTS™ instrument including disposable PTS™ cartridge FDA (0.05-5 EU/mL) (CRL). 2K-ET vial was directory exposed to mixed biogas in apparatus for 60 min at 37 or 60 degree celsius, and endotoxin concentrations were measured using 2K-ET samples dissolved in 1mL endotoxin-free water. After biogas exposure for 60 min., endotoxin concentration levels showed remarkably reduction up to 6∼11 %, and those effects suggested to be temperature dependent; 83.3±4.7 EU/mL at 37 degree, 46.0±0.85 EU/mL at 60 degree, control sample (non-exposure): 725±23.5 EU/mL. Thus, this innovative gas phase sterilization system could exhibit valuable potential as practical performance of endotoxin inactivation at low temperature as 37 degree. Although it is still necessary to develop the reduction performance into 3-Log reduction, this advantageous function is expected to be widely useful and have high potential in the public health and pharmaceutical industries in the future.

Validation and applicability of an alternative method for dialysis water and dialysate quality analysis.

ABSTRACTIntroduction:In hemodialysis, patients are exposed to a large volume of water, which may lead to fatal risks if not meeting quality standards. This study aimed to validate an alternative method for monitoring microbiological quality of treated water and assess its applicability in dialysis and dialysate analysis, to allow corrective actions in real-time.Methods:Validation and applicability were analyzed by conventional and alternative methods. For validation, E. coli standard endotoxin was diluted with apyrogenic water in five concentrations. For the applicability analysis, treated water for dialysis was collected from different points in the treatment system (reverse osmosis, drainage canalization at the storage tank bottom, reuse, and loop), and dialysate was collected from four machines located in different rooms in the hemodialysis sector.Results:The validation results were in accordance with the Brazilian Pharmacopoeia acceptance criteria, except for the last two concentrations analyzed. In addition, the ruggedness criterion performed under the US Pharmacopoeia was in agreement with the results.Discussion:A limiting factor in the applicability analysis was the absence of the endotoxin maximum permitted level in dialysate by the Brazilian legislation. When comparing the analysis time, the alternative method was more time-consuming than the conventional one. This suggests that the alternative method is effective in the case of few analyses, that is, real-time analyses, favoring corrective actions promptly. On the other hand, it does not support the implementation of the alternative method in a laboratory routine due to the high demand for analyses.

Proposed reduction of the in vivo pyrogen test by the in vitro LAL assay for the quality control of anticrotallic, antiscorpion, antirabies and antitetanus sera

The rationale for a formal study of the LAL (kinetic chromogenic method) assay for anticrotallic (SAC), antirabies (SAR), antitetanus (SAT) and antiscorpion (SAE) sera involved the determination of parameters required by the Brazilian National Health Surveillance Agency (ANVISA), the USA Food and Drug Administration (FDA), USP (United States Pharmacopeia, 39) and ICH (International Conference on Harmonization). The curve correlation coefficients obtained with the standard endotoxin control ranged from −0.980 to −1.000 in all experiments. Endotoxin recovery added to the SAC, SAR, SAT and SAE samples, at the working dilutions (1:10, 1:10, 1:10 and 1, 100 respectively), met the criteria required by the FDA, USP and Brazilian ANVISA for the Inhibition-Potentiation test. The applied methodology for the four analyzed sera fulfilled the required criteria for all performance parameters. Thus, the present study demonstrated that the in vivo pyrogen test can be potentially replaced by the LAL assay for all assessed sera samples displaying higher sensitivity and following the 3 Rs principle, in addition to maintaining quality control in Sanitary Surveillance.

Biophysical Analysis of Lipopolysaccharide Formulations for an Understanding of the Low Endotoxin Recovery (LER) Phenomenon.

Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called “Low Endotoxin Recovery (LER)”. LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration’s (FDA’s) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.

Detection of endotoxins using nanomaterials

An endotoxin is a lipopolysaccharide complex that is associated with the outer membrane of gram negative bacteria, and even a small amount can trigger strong exothermic activity. Pharmaceutical and food products can be contaminated with endotoxins, so there is a strict need to control them. A limulus amoebocyte lysate (LAL) assay is a standard endotoxin detection method that is highly sensitive and easy to use, but it has disadvantages in that the sample preparation is a complex process, it takes a long time to confirm the presence of endotoxins, and it requires a controlled workplace. Recently, endotoxin detection using nanomaterials has been gaining attention, and this review reports on the latest endotoxin detection methods using gold nanoparticles, magnetic nanoparticles, quantum dots, and carbon materials, among different nanomaterials, and it also introduces the future prospect of endotoxin detection.

Selective depletion of microglial progranulin in mice is not sufficient to cause neuronal ceroid lipofuscinosis or neuroinflammation

BackgroundProgranulin deficiency due to heterozygous null mutations in the GRN gene are a common cause of familial frontotemporal lobar degeneration (FTLD), while homozygous loss-of-function GRN mutations are thought to be a rare cause of neuronal ceroid lipofuscinosis (NCL). Aged progranulin-knockout (Grn-null) mice display highly exaggerated lipofuscinosis, microgliosis, and astrogliosis, as well as mild cell loss in specific brain regions. In the brain, progranulin is predominantly expressed in neurons and microglia, and previously, we demonstrated that neuronal-specific depletion of progranulin does not recapitulate the neuropathological phenotype of Grn-null mice. In this study, we evaluated whether selective depletion of progranulin expression in myeloid-lineage cells, including microglia, causes NCL-like neuropathology or neuroinflammation in mice.MethodsWe generated mice with progranulin depleted in myeloid-lineage cells by crossing mice homozygous for a floxed progranulin allele to mice expressing Cre recombinase under control of the LyzM promotor (Lyz-cKO).ResultsProgranulin expression was reduced by approximately 50–70% in isolated microglia compared to WT levels. Lyz-cKO mice aged to 12 months did not display any increase in lipofuscin deposition, microgliosis, or astrogliosis in the four brain regions examined, though increases were observed for many of these measures in Grn-null animals. To evaluate the functional effect of reduced progranulin expression in isolated microglia, primary cultures were stimulated with controlled standard endotoxin and cytokine release was measured. While Grn-null microglia display a hyper-inflammatory phenotype, Lyz-cKO and WT microglia secreted similar levels of inflammatory cytokines.ConclusionWe conclude that progranulin expression from either microglia or neurons is sufficient to prevent the development of NCL-like neuropathology in mice. Furthermore, microglia that are deficient for progranulin expression but isolated from a progranulin-rich environment have a normal inflammatory profile. Our results suggest that progranulin acts, at least partly, in a non-cell autonomous manner in the brain.

Biological Activity of Masked Endotoxin

Low endotoxin recovery (LER) is a recently discovered phenomenon describing the inability of limulus amebocyte lysate (LAL)-based assays to detect lipopolysaccharide (LPS) because of a “masking effect” caused by chelators or detergents commonly used in buffer formulations for medical products and recombinant proteins. This study investigates the masking capacities of different buffer formulations and whether masked endotoxin is biologically active. We show that both naturally occurring endotoxin as well as control standard endotoxin can be affected by LER. Furthermore, whereas masked endotoxin cannot be detected in Factor C based assays, it is still detectable in a cell-based TLR4-NF-κB-luciferase reporter gene assay. Moreover, in primary human monocytes, masked LPS induces the expression of pro-inflammatory cytokines and surface activation markers even at very low concentrations. We therefore conclude that masked LPS is a potent trigger of immune responses, which emphasizes the potential danger of masked LPS, as it may pose a health threat in pharmaceutical products or compromise experimental results.

Factors Affecting Reduction of Reference Endotoxin Standard Activity Caused by Chelating Agent/Detergent Matrices: Kinetic Analysis of Low Endotoxin Recovery.

Reduction of reference standard endotoxin activity was kinetically analyzed under low endotoxin recovery conditions and was considered as an apparent first-order reaction. Temperature, pH, and salt concentrations affected the rates of reduction of reference standard endotoxin activity. Temperature appeared to be the most important factor affecting low endotoxin recovery. Components of low endotoxin recovery matrices, such as citrate and polysorbate 20, showed similar low endotoxin recovery effect at concentrations commonly used. Phosphate concentrations showed negative correlation against the half-life of reference standard endotoxin activity in solutions containing phosphate buffer and polysorbate 20. Activation energy for low endotoxin recovery with naturally occurring endotoxin was higher than that with reference standard endotoxin, and this explained one of the reasons for naturally occurring endotoxin resistance to low endotoxin recovery. Lower temperature, lower pH, and a higher salt concentration are preferable to avoid low endotoxin recovery in a hold-time study. This study provides useful data for anticipation of the severity of the low endotoxin recovery effect and future hold-time studies in the biopharmaceutical field.LAY ABSTRACT: Endotoxin derived from Gram-negative bacteria is potentially harmful when it is parenterally administrated. Therefore, injectables and medical devices are tested by the bacterial endotoxins test to detect contamination by endotoxin of those products. Low endotoxin recovery is a phenomenon of reduction of detectable standard endotoxin activity by certain matrices of biopharmaceutical products containing a chelating agent and a detergent, and it is a controversial topic because its mechanism and clinical risks are unknown. The author analyzed the kinetics of low endotoxin recovery to elucidate the mechanism of low endotoxin recovery and to propose conditions to avoid low endotoxin recovery.

Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay

The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min−1 and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 °C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.

Molecular Imaging: Fundamentals and Applications

329 Objectives The pyrogen test represents an important parameter for QC of PET tracers [1]. However, the standard test proposed by the U.S. Pharmacopeia using limulus amebocyte lysates takes too long (about 1 h) [2] for short-lived PET tracers. Recently, a rapid test method, Endosafe® Portable Test System (PTS), has been approved by FDA and was claimed that it took only approximately 15 min to complete the test. The aims of this study were to evaluate PTSTM performance for PET tracers and head-to-head comparison with other traditional Limulus amebocyte lysate (LAL) systems. Methods PTS as well as traditional assay methods, Lonza kinetic chromogenic (KCA) and Endosafe® kinetic turbidimetric (KTA) assay systems were performed on 3 consecutive batches of on-site produced [18F]FDG, [11C]PiB and [13N]NH3 with 6 different executors (Fig. 1). The sensitivity of cartridges used for all samples tested with the PTS was 0.05-5.0 EU/mL. Additionally, fabricated endotoxin-positive samples containing a fixed concentration of endotoxin (RSE) were prepared and used as controls. Results The results of Endotoxin detection among these tripled methods were similar in most cases. However, the storage condition of endotoxin standards in KCA measurement has significant impact on the results of the Endotoxin detection and the KCA measurement is the most time-consuming method among these three methods ( the time of PTS, KCA and KTA were 16±1, 48±5 and 27± 1 min, respectively (n=9)). Conclusions The Endosafe® PTS provides reliable results for Endotoxin test and is the most suitable method for short-lived PET radiotracers.

Electrochemical sensor with substitutional stripping voltammetry for highly sensitive endotoxin assay.

We have developed a novel method for detection of endotoxin with extra-high sensitivity by using substitutional stripping voltammetry (SSV). In this method, a p-aminophenol (pAP) conjugated peptide (Boc-Leu-Gly-Arg-pAP; LGR-pAP) was used as a substrate for a protease, which is activated at the last step of the endotoxin-induced Limulus amebocyte lysate (LAL) cascade reaction. Extra-highly sensitive detection of pAP liberated by the endotoxin-induced LAL reaction was successfully realized with SSV, based on the accumulation of an amperometric signal owing to exchange of the oxidation current of pAP generated at an electrode in a reaction cell with silver deposition on another electrode in a deposition cell. This reaction is driven by the difference in the redox potential between pAP/quinoneimine and silver/silver ion. The amount of the deposited silver is quantified by anodic stripping voltammetry (ASV). This SSV-based endotoxin assay was performed with a chip device comprising two cells, each of which was connected via a liquid junction made of Vycor® glass. The reaction cell and the deposition cell contained a standard endotoxin sample with LAL regents containing LGR-pAP and AgNO3 solution, respectively. After the cells were electrically connected for 60 min, ASV was conducted in the deposition cell to quantify the total electrical charge derived by the oxidation of free pAP in the reaction cell. The ASV signal increased with the increase of the endotoxin concentration in the sample solution in the range of 0.5-1000 EU L(-1).

92. Control standard endotoxin as a potential tool to reduce the variability observed with lipopolysaccharide

Peripheral administration of the endotoxin lipopolysaccharide (LPS) is a commonly used immunological challenge in animal models of inflammation-associated depressive-like behavior. LPS stimulates the production of pro-inflammatory cytokines, leading to an induction of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO). Elevated IDO activity, as measured by the dysregulation of kynurenine pathway metabolites, is proposed to mediate inflammation-induced depressive-like behavior in mice. LPS is subject to considerable variability in potency, which can often lead to variability in results. Control standard endotoxin (CSE) is a highly purified LPS preparation and may reduce the variability in results observed with LPS. CD-1 mice were treated with 0, 0.15, 1.5, 15 or 150 μg/kg CSE i.p. and sacrificed 24 h later. Brain and plasma were collected for analysis of cytokines and kynurenine pathway metabolites. The highest dose of CSE (150 μg/kg) increased kynurenine, which was metabolized towards the quinolinic branch of the kynurenine pathway in the brain and plasma. The magnitude of change in kynurenine metabolites was similar to those found in previous studies with 1 mg/kg LPS. Mice treated with CSE had higher levels of interleukin (IL)-1 beta and tumor necrosis factor alpha in the brain, and IL-10 and interferon gamma in the plasma. These results suggest that CSE may be used as an improvement over LPS in an in vivo neuroinflammation model.