Limulus Amoebocyte Lysate Test Research Articles

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.

Endotoxin Contamination and Reaction Interfering Substances in the Plant Extract Library.

Endotoxin is an unintentional contaminant that has numerous activities and can affect various biological experiments using cells. In this study, we measured the endotoxin activity of samples from a plant extract library (PEL) and determined their degrees of contamination. Endotoxin was detected in approx. 48% (n = 139) and approx. 4% (n = 5) of field-collected and crude drug samples, respectively, and in concentrations >5.0 EU/mL in some samples. The concentrations of endotoxin that affect cells in vitro vary depending on the target cell type. Although the degree of contamination varied in the present study, it was considered to have little effect on the cell experiments. More than 150 PEL samples had problems with reaction courses or recovery rates of Limulus amoebocyte lysate (LAL) tests. In the LAL tests, using three plant extracts [Sanguisorba officinalis L. (Rosaceae), Oenothera biennis L. (Onagraceae), and Lythrum salicaria L. (Lythraceae)], the polyphenolic compounds in the plant extracts affected LAL test and their effects differed depending on the plant species. When the 16 single polyphenol compounds were added to the LAL tests, the compounds with caffeoyl and pyrogallol moieties were found to affect the LAL reaction course and recovery rate. Furthermore, none of the compounds had any effects at concentrations of 1 µM. Because the plant extracts contained analogs of various polyphenolic compounds, they were presumed to actually act synergistically. Our findings demonstrated that attention must be paid to the recovery rate and reaction process of LAL tests with samples containing polyphenolic compounds.

A possible beneficial effect of Bacteroides on faecal lipopolysaccharide activity and cardiovascular diseases

Faecal lipopolysaccharides (LPS) have attracted attention as potent elements to explain a correlation between the gut microbiota and cardiovascular disease (CVD) progression. However, the underlying mechanism of how specific gut bacteria contribute to faecal LPS levels remains unclear. We retrospectively analysed the data of 92 patients and found that the abundance of the genus Bacteroides was significantly and negatively correlated with faecal LPS levels. The controls showed a higher abundance of Bacteroides than that in the patients with CVD. The endotoxin units of the Bacteroides LPS, as determined by the limulus amoebocyte lysate (LAL) tests, were drastically lower than those of the Escherichia coli LPS; similarly, the Bacteroides LPS induced relatively low levels of pro-inflammatory cytokine production and did not induce sepsis in mice. Fermenting patient faecal samples in a single-batch fermentation system with Bacteroides probiotics led to a significant increase in the Bacteroides abundance, suggesting that the human gut microbiota could be manipulated toward decreasing the faecal LPS levels. In the clinical perspective, Bacteroides decrease faecal LPS levels because of their reduced LAL activity; therefore, increasing Bacteroides abundance might serve as a novel therapeutic approach to prevent CVD via reducing faecal LPS levels and suppressing immune responses.

Endotoxin quality control testing for CAR T-cell manufacturing: validation considerations for Endosafe portable testing system

Background & Aim Background Adoptive cell therapies have emerged as promising treatments for cancer patients. The manufacture of clinical-grade Chimeric Antigen Receptor (CAR) T-cells therapies encompass multiple complex processes under the code of good manufacturing practice (cGMP) to ensure high-quality and safe products. Endotoxins are lipo-polysaccharides from gram-negative bacteria that cause serious adverse events in patients. For this reason, it is important that CAR T-cells are tested for endotoxin content. The limulus amoebocyte lysate (LAL) test is FDA approved and allows sensitive detection of bacterial endotoxins. The Endosafe Portable Testing System (PTS) is a rapid, FDA approved testing system manufactured by Charles River that uses disposable cartridges for accurate real-time endotoxin testing, However, a number of important parameters need to be considered when establishing Endosafe-PTS testing for CAR T-cells products. Aim To determine endotoxin limits, maximum valid dilution of samples and reproducibility of the Endosafe-PTS test. Methods, Results & Conclusion Methods Inhibition and enhancement screening cartridges were used initially to establish maximum valid dilution and investigate potential inhibition of the test. CAR T-cells were manufactured from three individuals and analysed for endotoxins on the Endosafe-PTS using cartridges with a sensitivity of 5-0.05 EU/mL and spike recovery 50-200% according to manufacturer's protocol. Three replicates of each sample were tested. Results The optimal dilution of CAR T-cell product was determined at 1:20 with the spike recovery of 84% to 130%. No inhibition was detected at the 1:20 dilution for all three samples. The upper limit of acceptance criteria for endotoxin was determined on the basis of dose and FDA guidelines to be 5 EU/kg body weight/hour of intended product administration. Conclusion The Endosafe-PTS provides an accurate, reproducible and rapid method for Endotoxin detection which is crucial in quality control release testing and administration of time-sensitive CAR T-cell products. It is important to validate inhibition and enhancement screening for each CAR T-cell product and determine the optimal sample dilution before implementation of the Endosafe-PTS for product release testing.

Extraction and Evaluation of Outer Membrane Vesicles from Two Important Gut Microbiota Members, Bacteroides fragilis and Bacteroides thetaiotaomicron.

Objective The gastrointestinal tract (GI) is colonized by a complex microbial community of gut microbiota. Bacteroides spp. have significant roles in gut microbiota and they host interactions by various mechanisms, including outer membrane vesicle (OMVs) production. In the present study, we extracted and assessed Bacteroides fragilis (B. fragilis) and Bacteroides thetaiotaomicron (B. thetaiotaomicron) OMVs in order to evaluate their possible utility for in vivo studies. Materials and Methods In this experimental study, OMVs extraction was performed using multiple centrifugations and tris-ethylenediaminetetraacetic acid (EDTA)-sodium deoxycholate buffers. Morphology, diameter, protein content, profile, and lipopolysaccharide (LPS) concentrations of the OMVs were assessed by scanning electron microscopy (SEM), nanodrop, Bradford assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the Limulus Amoebocyte Lysate (LAL) test, respectively. Zeta potential (ζ-P) was also assessed. The viability effect of OMVs was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay in Caco-2 cells. Results Spherical OMVs with diameters of 30-110 nm were produced. The OMVs had different protein profiles. The LPS concentrations of the B. fragilis and B. thetaiotaomicron OMVs were 1.80 and 1.68 EU/mL, respectively. ζ-P of the B. fragilis OMVs was -34.2 mV and, for B. thetaiotaomicron. it was -44.7 mV. The viability of Caco-2 cells treated with OMVs was more than 95%. ConclusionThe endotoxin concentrations of the spherical OMVs from B. fragilis and B. thetaiotaomicron were within the safe limits. Both OMVs had suitable stability in sucrose solution and did not have any cytotoxic effects on human intestinal cells. Based on our results and previous studies, further molecular evaluations can be undertaken to design OMVs as possible agents that promote health properties.

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.

Bacterial contamination hypothesis: a new concept in endometriosis.

BackgroundEndometriosis is a multifactorial disease that mainly affects women of reproductive age. The exact pathogenesis of this disease is still debatable. The role of bacterial endotoxin (lipopolysaccharide, LPS) and Toll‐like receptor 4 (TLR4) in endometriosis were investigated and the possible source of endotoxin in the pelvic environment was examined.MethodsThe limulus amoebocyte lysate test was used to measure the endotoxin levels in the menstrual fluid and peritoneal fluid and their potential role in the growth of endometriosis was investigated. Menstrual blood and endometrial samples were cultured for the presence of microbes. The effect of gonadotrophin‐releasing hormone agonist (GnRHa) treatment on intrauterine microbial colonization (IUMC) and the occurrence of endometritis was investigated.Main findings (Results)Lipopolysaccharide regulates the pro‐inflammatory response in the pelvis and growth of endometriosis via the LPS/TLR4 cascade. The menstrual blood was highly contaminated with Escherichea coli and the endometrial samples were colonized with other microbes. A cross‐talk between inflammation and ovarian steroids or the stress reaction also was observed in the pelvis. Treatment with GnRHa further worsens intrauterine microbial colonization, with the consequent occurrence of endometritis in women with endometriosis.ConclusionFor the first time, a new concept called the “bacterial contamination hypothesis” is proposed in endometriosis. This study's findings of IUMC in women with endometriosis could hold new therapeutic potential in addition to the conventional estrogen‐suppressing agent.

Platelets: an outlook from biology through evidence-based achievements in critical care.

Since the original observations by Bizzozero and Osler, we have seen tremendous advances in the understanding of platelets far beyond haemostasis and the restoration of injured endothelium. In this mini-review on platelets, we will briefly outline their historical description and the importance of their evolution, focusing on a 450 million years old living fossil of Limulus polyphemus, a marine chelicerate arthropod, which helped researchers explain the basis for the immunity role of platelets and make correlations with platelet ultrastructure and function. In addition, the impact of the Limulus Amoebocyte Lysate (LAL) test for modern medicine is highlighted. The role of platelets in cardiovascular diseases, their relevance in arterial and venous thrombosis, and the utilization of antithrombotic drugs as therapeutic agents are also reported. Furthermore, platelet receptors are crucial in aggravating or mitigating other diseases, such as cancer and infections, which can recruit cells and have numerous interactions in a process recently coined "NETosis formation", which is also briefly depicted.

More than 70 years of pyrogen detection: Current state and future perspectives.

In the quality assurance of medical products, tests for sterility are essential. For parenteral pharmaceuticals, avoiding the presence of pyrogens is crucial. These fever-inducing substances (endotoxins and non-endotoxins) are not eliminated by standard sterilisation processes, and are biologically active once in the bloodstream, causing risks to human health, ranging from mild reactions (e.g. fever) to septic shock and death. Therefore, for injectable formulations, pyrogen testing is mandatory. Over the years, various pyrogen testing methods have been introduced, namely: in the 1940s, the rabbit pyrogen test, which is an in vivo test that measures the fever reaction as an endpoint; in the 1970s, the Limulus Amoebocyte Lysate (LAL) test, which is an in vitro test (with the haemolymph of the horseshoe crab) that specifically detects endotoxin; and in 2010, the Monocyte-Activation Test (MAT), which is a non-animal based in vitro pyrogen test that represents a full replacement of the rabbit test. Due to the ubiquity and biological significance of pyrogens, we are currently further developing the MAT so that it can be used for other applications. More specifically, our focus is on the detection of pyrogenic contamination on medical devices, as well as on the measurement of air quality. In addition, further improvements to permit the use of cryopreserved blood in the MAT, to overcome the limitations in the availability of freshly-drawn blood from human donors, are ongoing.

Lipopolysaccharide quantification and alkali-based inactivation in polysaccharide preparations to enable in vitro immune modulatory studies

The correct identification of immune-modulatory activity of polysaccharides is often hampered by immune-stimulatory contaminants, with pyrogens such as lipopolysaccharide (LPS) as a very potent example. In order to avoid false positive immuno-stimulatory properties to be attributed to polysaccharides, accurate quantification and inactivation of LPS in test samples is crucial. To quantify LPS in polysaccharide preparations of different origin and structure we used two different limulus amoebocyte lysate test kits in two different laboratories. We observed larger variation in detection of LPS contamination between kits than between labs. LPS quantification proved unreliable for some polysaccharide preparations as spike controls resulted in spike recoveries outside the acceptable range. We designed a cellular in vitro assay as alternative method to detect the presence of functional LPS. This HEK-Blue hTLR4 cell culture provides a reliable assay, when combined with a cell viability test, for determining functional LPS in polysaccharide preparations. Finally, to inactivate LPS in polysaccharide preparations, we setup an alkaline-ethanol-based treatment. With this assay we observed that our treatment (5h incubation in 0.1M NaOH) at 56°C efficiently inactivated LPS in all polysaccharide preparations below immune-stimulatory levels. At this elevated temperature, however, we also observed minimal to severe degradation of polysaccharide preparations as determined with SEC-RI. Taken together, we describe methods and precautions to reliably detect and inactivate LPS in polysaccharide preparations to allow reliable in vitro investigations towards immune-modulatory potential of polysaccharide preparations.

Lipopolysaccharide (LPS) and Protein-LPS complexes: Detection and Characterization by Gel Electrophoresis, Mass Spectrometry and Bioassays

A prerequisite to the discovery and characterization of lipopolysaccharide (LPS) interaction with specific receptors and the resulting pathophysiological effects is the comprehensive structural analysis of LPS species. This brief review is aimed to summarize the use of gel electrophoresis linked to other biochemical technologies for detecting and characterizing LPSs. Lipopolysaccharide aggregates alone or mixtures containing LPSs and proteins/peptides can be separated by native agarose gel electrophoresis (NAGE), after which LPSs are detected with imidazole and zinc salts. A double-staining process with Coomassie brilliant blue R-250 enables the use of NAGE for detecting and studying protein-LPS interactions. For compositional analysis, the LPS aggregates are separated, with high resolution, by surfactant-polyacrylamide gel electrophoresis. After reverse staining with zinc-imidazole and elution from gel microparticles, glycoform-specific LPSs are ready for structural and biological analysis. For sequence analysis based on tandem electrospray ionization mass spectrometry (ESI-MS/MS) of oligosaccharides, LPSs are subjected to mild acid hydrolysis, dephosphorylation and permethylation. Also, O-deacylated LPS forms can be analyzed by matrix-assisted laser desorption/ionization-time of flight-MS. By comparison to spectra of unpurified LPSs, mass spectra of the micropurified LPSs show reduce heterogeneity and increased signal-to-noise ratios. Furthermore, the micropurification of LPSs prior to MS allows a higher sensitivity of detection for less abundant LPS glycoforms. The micropurified LPS fractions can be used to form self-assembled nanoaggregates which may be detected by dynamic light scattering. The effect of the O-side chain length on the Z-potential of LPS aggregates may be estimated by measurements based on laser Doppler electrophoresis. The thus obtained glycoform-specific LPSs are not only intact chemically but also biologically active as tested by e.g. Limulus amoebocyte lysate test, TNF-α assay and agonistic effect on human Toll-like receptor 4.

The interaction of human macrophage subsets with silicone as a biomaterial

Silicones are widely used as biomaterials for medical devices such as extracorporeal equipments. However, there is often conflicting evidence about their supposed cell- and histocompatibility. Macrophages could mediate silicone-induced adverse responses such as foreign body reaction and fibrous encapsulation. The polarization behaviour of macrophages could determine the clinical outcome after implantation of biomaterials. Induction of classically activated macrophages (CAM) may induce and support uncontrolled inflammatory responses and undesired material degradation. In contrast, polarization into alternatively activated macrophages (AAM) is assumed to support healing processes and implant integration.This study compared the interaction of non-polarized macrophages (M0), CAM, and AAM with commercially available tissue culture polystyrene (TCP) and a medical grade silicone-based biomaterial, regarding the secretion of inflammatory mediators such as cytokines and chemokines. Firstly, by using the Limulus amoebocyte lysate (LAL) test the silicone films were shown to be free of soluble endotoxins, which is the prerequisite to investigate their interaction with primary immune cells. Primary human monocyte-derived macrophages (M0) were polarized into CAM and AAM by addition of suitable differentiation factors. These macrophage subsets were incubated on the materials for 24 hours and their viability and cytokine secretion was assessed. In comparison to TCP, cell adhesion was lower on silicone after 24 hours for all three macrophage subsets. However, compared to TCP, silicone induced higher levels of certain inflammatory and chemotactic cytokines in M0, CAM, and AAM macrophage subsets.Conclusively, it was shown that silicone has the ability to induce a pro-inflammatory state to different magnitudes dependent on the macrophage subsets. This priming of the macrophage phenotype by silicone could explain the incidence of severe foreign body complications observed in vivo.

Methods of Endotoxin Detection.

Endotoxin, present in the outer membrane of all gram-negative bacteria, can pose serious risks to human health, from irreversible shock to death. Therefore, it is essential to develop sensitive, accurate, and rapid methods for its detection. The rabbit pyrogen test is the first standard technique for endotoxin detection and, nowadays, has been replaced by the Limulus Amoebocyte Lysate test, which is the most popular detection technique for endotoxin. With in-depth understanding of endotoxin, biosensors based on endotoxin-sensing components are promising alternatives to pursue in developing low-cost, easy-operation, and fast-response endotoxin detection techniques. This article summarizes the recent advances of endotoxin detection methods with a particular emphasis on optical and electrochemical biosensors based on various sensing elements ranging from nature biomolecules to artificial materials. As the research and technological revolution continues, the highly integrated and miniaturized commercial devices for sensitively and reliably detecting endotoxin will provide a wide range of applications in people's daily life.

Dust, endotoxin, fungi, and bacteria exposure as determined by work task, season, and type of plant in a flower greenhouse.

Greenhouse workers are exposed to dust, endotoxin, fungi, and bacteria potentially causing airway inflammation as well as systemic symptoms. Knowledge about determinants of exposure is a prerequisite for efficient prevention through knowledge-based reduction in exposure. The objective of this study was to assess the occupational exposure in a flower greenhouse and to investigate the impact of work tasks on the intensity and variability in exposure. Seventy-six personal full-shift exposure measurements were performed on 38 employees in a Danish flower greenhouse producing Campanula, Lavandula, Rhipsalideae, and Helleborus. The samples were gravimetrically analysed for inhalable dust. Endotoxin was assessed by the Limulus Amoebocyte Lysate test and culture-based quantification of bacteria and fungi was performed. Information on the performed tasks during sampling was extracted from the greenhouse electronic task logging system. Associations between log-transformed exposure outcomes, season, and work tasks were examined in linear mixed-effects regression with worker identity as random effect. Measured concentrations ranged between 0.04 and 2.41mg m(-3) for inhalable dust and between 0.84 and 1097 EU m(-3) for endotoxin exposure, with the highest mean levels measured during Lavandula and Campanula handling, respectively. Personal exposure to fungi ranged between 1.8×10(2) and 3.4×10(6) colony-forming units (CFU) m(-3) and to bacteria between 1.6×10(1) and 4.2×10(5) CFU m(-3). Exposure to dust, endotoxin, fungi, and bacteria differed between seasons. Packing Lavandula, sticking, potting, and grading Rhipsalideae, and all examined tasks related to Campanula production except sticking increased dust exposure. Endotoxin exposure was increased during sticking Campanula and pinching or packing Rhipsalideae, and fungi exposure was elevated by subtasks performed in the research and development area for Campanula, and by potting, packing/dumping Campanula. Sticking and working with subtasks in the research and development area for Campanula increased bacteria exposure. This study revealed moderate dust exposure levels compared to the levels observed in other greenhouse productions and other occupations with organic dust exposure such as farming. However, high exposures to bacteria and fungi were detected during selected tasks and the proposed health-based endotoxin exposure limit of 90 EU m(-3) was exceeded in 30% of the samples, which may have health implications for the employees. Exposure levels were found to vary depending on the tasks performed, and thereby results can be used to direct task-based initiatives to reduce workplace exposures.

Prostaglandin E2 as a read out for endotoxin detection in a bovine whole blood assay.

The detection of endotoxin contamination is an essential part of drug safety testing. The rabbit pyrogen test (RPT), the limulus amoebocyte lysate (LAL) test, and the monocyte activation test (MAT) are established methods for the detection of pyrogens. However, the RPT is insufficiently standardized; the LAL test is solely capable of identifying the presence of endotoxins, whereas the use of the MAT is limited by the availability of human blood. Here, we introduce a new procedure for testing endotoxin contamination using prostaglandin E2 (PGE2 ) release from bovine whole blood. We incubated bovine whole blood overnight with lipopolysaccharide (LPS) from Escherichia coli 0111:B4, concentrations ranging from 1.56 to 12.5pg/mL, and found significantly increased PGE2 production for even the lowest LPS concentrations. Testing the possibility of storing the blood at 4°C before use also yielded positive results as 1.56pg/mL still significantly increased PGE2 production, thus suggesting some flexibility of the assay regarding time. These results emphasize the potential of using bovine whole blood for highly sensitive endotoxin testing. As a perspective, currently ongoing research aims to show whether the assay is also capable of detecting nonendotoxin pyrogens.

Choice of Method for Endotoxin Detection Depends on Nanoformulation

Many nanoparticles interfere with traditional tests to quantify endotoxin. The aim of this study was to compare the performance of limulus amoebocyte lysate (LAL) formats on clinical-grade nanoformulations, to determine whether there were disparate results among formats and to test the applicability of an alternative bioassay (the macrophage activation test [MAT]) for resolving discrepancies, if observed. Clinical-grade nanoformulations were tested using turbidimetric, gel-clot and chromogenic LAL. Formulations that cause a discrepancy among LAL tests were also tested by the MAT. The gel-clot LAL method cannot be relied upon to resolve discrepancies among LAL tests for certain nanoformulations. No one LAL format was shown to be optimal for all the tested clinical-grade nanoformulations. The tested alternative bioassay (the MAT) was useful for verifying LAL findings, but only for those nanoformulations not carrying/including cytotoxic drugs.

Series of fibrinous inflammation after implantation of capsular tension rings

To analyze the cause of fibrinous inflammation in eyes with capsular tension rings (CTRs) after cataract surgery. Department of Ophthalmology, University Medical Center, Johannes Gutenberg-University Mainz, Mainz, Germany. Retrospective case series. High-performance liquid chromatography was implemented to eliminate impurities. One CTR was explanted for microbiologic examination. The pH values of the CTR and the storage solution were analyzed, Seldi and Maldi tests were performed, as well as toxicity tests with immortal cell lines. Material batches were analyzed. The organic carbon content of CTRs, detergents, and storage solutions was checked. The presence of endotoxins was excluded with the limulus amoebocyte lysate test. Gas chromatography with mass selective detector excluded the presence of extractable organic substances. An inductively coupled plasma analysis scanned for inorganic substances. The microbial count in operating rooms, smear tests, and microbiologic examinations of technical devices were initiated. Analyses found no pathological findings. After intensive systemic and topical treatment with antibiotics and steroids, clinical findings improved. The implantation of CTRs was stopped immediately. No further cases occurred. Fibrinous inflammations after implantation of CTRs were unknown until now. Contamination and/or irritation by detergents or the material CTR itself were excluded, indicating mechanical or toxic irritation by the CTR. No author has a financial or proprietary interest in any material or method mentioned.

Stability and purity of a bacteriophage cocktail preparation for nebulizer delivery

The aim of this study was to determine the stability and purity of a phage cocktail to be delivered by nebulization. A cocktail of three phages active against Pseudomonas aeruginosa isolates from cystic fibrosis patients was developed for a potential nebulized formulation. The individual phages were examined for their retention of activity over time, while the phage cocktail was analysed for bacterial contaminant and endotoxin level according to regulatory requirements for nebulized products. The phage cocktail was nebulized using a Porta-neb nebulizer connected to an Anderson cascade impactor. The three phages retained activity over a period of 180 days storage at room temperature and at 4°C. Nebulized phages were recovered in the lower stages of the cascade impactor indicative of potential delivery deep into the lungs. The phage cocktail met bacterial limits but the endotoxin levels measured with the Limulus amoebocyte lysate (LAL) test remained considerably in excess of acceptable levels even following purification. These findings suggest that nebulization of phage is a viable delivery option; although, there is a need for appropriate depyrogenation strategies to remove bacterial endotoxins from phage-based preparations to meet regulatory requirements. With increasing reports of bacterial resistance to antibiotics and the lack of new antibiotics being produced, bacteriophage therapy is becoming an attractive alternative. There has been no published report on the quality assurance of bacteriophage product to date. This is the first study on the quality assurance of a Pseudomonas aeruginosa phage cocktail following pharmacopoeial requirements. The presence of bacterial endotoxin was found to be the key stumbling block for meeting regulatory criteria.

Environmental and occupational respiratory diseases – 1042. Influence of environmental endotoxin and allergen levels on prevalence of asthma and allergen sensitizations in urban and rural children in Guandong, China

Methods Three hundred and seventeen children aged 13-14 years (Guangzhou 188, Conghua 129) with self-reported respiratory symptoms and 537 healthy children were selected from 854 school-children according to their response to ISAAC standardized questionnaire. These children attended the school infirmary again to complete a detailed questionnaire and medical examinations including skin prick test (SPT) to 8 common aeroallergens, lung function test, histamine bronchial provocation test and peripheral blood eosinophil counts. House dust samples were collected from living room and mattresses of 156 of these children with the agreement of their parents (Guangzhou 76, Conghua 80). The content of endotoxin in house dust was quantified using an endpoint chromogenic kinetic limulus amoebocyte lysate test. Associations between levels of house dust allergens and endotoxin and the provocation dose producing a 20% fall in forced expiratory volume in one second (PD20-FEV1) and allergen skin reactions were analysed by logistic regression. Results The prevalence of wheeze symptom, asthma, nasal symptoms and allergic rhinitis in the children of Guangzhou were significantly higher (28.6%, 27.7%, 66%, 46.4%, respectively) than the children of Conghua (5%, 2.5%, 31%, 6.2%, respectively, p<0.01). Endotoxin concentrations in dust samples in homes of Conghua were significantly higher (1794 EU per square meter, 10.95 EU endotoxin/g dust) than those of Guangzhou (508.8 EU per square meter, 6.45 EU endotoxin/g dust, p<0.01). Logistic regression analyses showed a positive association between endotoxin levels and PD20-FEV1 (r=0.174, p<0.05), and a negative association (p<0.01) between endotoxin levels and the wheal sizes of skin response to Dermatophagoides pteronyssinus (Der. p) and Dermatophagoides farinae (Der. f).

Detection of Endotoxin Concentration Using Piezoelectric Based Biosensor System

Endotoxins are ubiquitous in the environment and represent important pathogenic molecules. In this paper, we present a sensitive and reliable method for quantitation and detection of endotoxin based on piezoelectric biosensors, which monitor the gel formation (causing viscosity change) when Limulus Amoebocyte Lysate (LAL) mixed with endotoxin. The resonance frequency shifts of the sensors in contact with different concentration endotoxin were recorded as a function of time. Results showed that there has good relationship between the logarithmic concentration of endotoxin and the maximum clotting rate as well as the corresponding clotting time. The detection limits could be gone down to 0.1pg/ml and the time-consuming is about 1 hour using this system. By comparison of the traditional methods (rabbit pyrogen test and LAL test) for detection of endotoxin, the proposed sensor is much simpler, more precise and lower detection limits.