Limulus Amebocyte Lysate Gel Clot Research Articles

A method to prevail false positive responses due to excess cations and viscous nature of Radiopharmaceuticals in Limulus Amebocyte Lysate Gel Clot test

Objective(s):Bacterial endotoxin test (BET) for detection and quantification of endotoxin in radiopharmaceuticals (RPs), used for therapy or diagnosis, is prerequisite to administration in patients. Out of the two established methods used for this purpose (Kinetic Chromogenic Assay: KCM and Gel Clot Bacterial Endotoxin Test: GC-BET), GC-BET is recommended by pharmacopeias to evaluate the interferences exhibited during the assay due to presence of various ingredients in samples. In the present study, the influence of excess of cations in [177Lu]Lu-DOTATATE, used for Peptide Receptor Radionuclide Therapy (PRRT), were studied and a protocol to negate the enhancement observed was developed. Additionally, a protocol for carrying out GC-BET for extremely viscous [131I]I-Lipiodol was standardized.Methods:GC-BET was performed for [177Lu]Lu-DOTATATE and [131I]I-Lipiodol at maximum valid dilution (MVD), using LRW as a diluent. To negate the false positivity observed in case of [177Lu]Lu-DOTATATE, various concentrations of calcium chloride (CaCl2) were added and evaluated for the reversal of the interference observed initially. To prevail the difficulty in performing GC-BET for [131I]I-Lipiodol various modification in the protocols like orbital vortexing at different rpm and time intervals were performed. KCM assays were also performed for studied RPs at MVD.Results:It was observed that at MVD, [177Lu]Lu-DOTATATE exhibited false positivity in GC-BET. However, all the individual reagents used in labeling of [177Lu]Lu-DOTATATE did not show any false positivity. Finally, performing the assay with an addition of 2mM CaCl2 (final concentration) nullified the false positivity. Further, intricacy in performing GC-BET for [131I]I-Lipiodol due to its viscosity was resolved by orbital vortexing at 3000 rpm for 5 minutes.Conclusions:Our study proved that false positivity was observed in GC-BET for [177Lu]Lu-DOTATATE due to the presence excess M3+ ions. Further, our study is the first of its kind which demonstrated methods for negating these false positive results by using modified protocol and hypothesizing the reason behind the enhancement. Additionally, ours is the first study which proved that a simple step of vortexing the viscous RPs like [131I]I-Lipiodol can resolved the problems encountered during performing GC-BET due to viscosity of RPs.

Interference of Gold Nanoparticles with In vitro Endotoxin Detection Assays

Background: Endotoxin-free engineered nanoparticle suspensions are imperative for their successful applications in the field of nanomedicine as well as in the investigations in their toxicity. Gold nanoparticles are known to interfere with various in vitro assays due to their optical properties and potential for surface reactivity. In vitro endotoxin testing assays are known to be susceptible to interference caused by the sample being tested. Objective: This study aimed to identify a preferred assay for the testing of endotoxin contamination in gold nanoparticle suspensions. Methods: The interference by gold nanoparticles on three assays namely, the commonly used limulus amebocyte lysate chromogenic assay, the limulus amebocyte lysate gel-clot method, and the less common recombinant Factor C (rFC) assay, was tested. Results: Possible interference could be observed with all three assays. The interference with the absorbance- based chromogenic assay could not be overcome by dilution; whilst the qualitative nature of the gel-clot assay excluded the possibility of distinguishing between a false positive result due to enhancement of the sensitivity of the assay, and genuine endotoxin contamination. However, interference with the rFC assay was easily overcome through dilution. Conclusion: The rFC assay is recommended as an option for endotoxin contamination detection in gold nanoparticle suspensions.

Bacteria and endotoxin in meconium-stained amniotic fluid at term: could intra-amniotic infection cause meconium passage?

Background: Meconium-stained amniotic fluid (MSAF) is a common occurrence among women in spontaneous labor at term, and has been associated with adverse outcomes in both mother and neonate. MSAF is a risk factor for microbial invasion of the amniotic cavity (MIAC) and preterm birth among women with preterm labor and intact membranes. We now report the frequency of MIAC and the presence of bacterial endotoxin in the amniotic fluid of patients with MSAF at term.Materials and methods: We conducted a cross-sectional study including women in presumed preterm labor because of uncertain dates who underwent amniocentesis, and were later determined to be at term (n = 108). Patients were allocated into two groups: (1) MSAF (n = 66) and (2) clear amniotic fluid (n = 42). The presence of bacteria was determined by microbiologic techniques, and endotoxin was detected using the Limulus amebocyte lysate (LAL) gel clot assay. Statistical analyses were performed to test for normality and bivariate comparisons.Results: Bacteria were more frequently present in patients with MSAF compared to those with clear amniotic fluid [19.6% (13/66) versus 4.7% (2/42); p < 0.05]. The microorganisms were Gram-negative rods (n = 7), Ureaplasma urealyticum (n = 4), Gram-positive rods (n = 2) and Mycoplasma hominis (n = 1). The LAL gel clot assay was positive in 46.9% (31/66) of patients with MSAF, and in 4.7% (2/42) of those with clear amniotic fluid (p < 0.001). After heat treatment, the frequency of a positive LAL gel clot assay remained higher in the MSAF group [18.1% (12/66) versus 2.3% (1/42), p < 0.05]. Median amniotic fluid IL-6 concentration (ng/mL) was higher [1.3 (0.7–1.9) versus 0.6 (0.3–1.2), p = 0.04], and median amniotic fluid glucose concentration (mg/dL) was lower [6 (0–8.9) versus 9 (7.4–12.6), p < 0.001] in the MSAF group, than in those with clear amniotic fluid.Conclusion: MSAF at term was associated with an increased incidence of MIAC. The index of suspicion for an infection-related process in postpartum women and their neonates should be increased in the presence of MSAF.

Nanoparticle interactions on a cellular scale

Nanoobjects are distinguished from other materials by their nanoscale size. The special physico-chemical properties of nanoobjects attributed to size-effects are the basis for their high application potential. Some of these properties are considered to be the basis for biologically relevant effects of nanoobjects. These are promoted by a) specific entry pathways and distribution within the body and tissues, b) uptake into single cells and specific intracellular distribution, and c) a high chemical reactivity, influencing dissolution, adsorption patterns and kinetics, or agglomeration. To take this even further, the composition and morphology of nanoobjects, their surface properties as well as the composition of the surrounding media influence the interactions of nanoobjects with molecules and living entities. Focussing on the intracellular location of nanoparticles, we are using well-defined and fluorescently labelled nanoparticles. In order to detect cell-associated 25 – 130nm silica particles, confocal and the recently developed STED (STimulated Emission Depletion) microscopy are applied. An introduction into the latter technique, allowing for sub-diffraction resolution, is given and results of microscopic studies are presented. Furthermore, the location of 3 – 15nm gold nanoparticles after uptake into A549 cells as model for type II alveolar cells was studied using light and correlative light and electron microscopy. The distribution pattern of 3nm gold nanoparticles seemed to be different compared with larger nanoparticles. At relevant concentrations, particles induced changes of the cellular morphology, involving the cytoskeleton. Biochemical assays are generally used to analyse nanoparticle effects. Nanotoxicity studies using conventional assays are prone to interference of the particles with the test systems. As an example for direct effects of nanoparticles on such assays, the interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay was analysed. In the presence of particles, not only inhibition, but also enhancement of the test system was observed. The influence of nanoparticle properties on biological assays is discussed.

Interference of silica nanoparticles with the traditional Limulus amebocyte lysate gel clot assay

Endotoxin contaminations of engineered nanomaterials can be responsible for observed biological responses, especially for misleading results in in vitro test systems, as well as in vivo studies. Therefore, endotoxin testing of nanomaterials is necessary to benchmark their influence on cells. Here, we tested the traditional Limulus amebocyte lysate gel clot assay for the detection of endotoxins in nanoparticle suspensions with a focus on possible interference of the particles with the test system. We systematically investigated the effects of nanomaterials made of, or covered by, the same material. Different types of bare or PEGylated silica nanoparticles, as well as iron oxide-silica core shell nanoparticles, were tested. Detailed inhibition/enhancement controls revealed enhanced activity in the Limulus coagulation cascade for all particles with bare silica surface. In comparison, PEGylation led to a lower degree of enhancement. These results indicate that the protein-particle interactions are the basis for the observed inhibition and enhancement effects. The enhancement activity of a particle type was positively related to the calculated particle surface area. For most silica particles tested, a dilution of the sample within the maximum valid dilution was sufficient to overcome non-valid enhancement, enabling semi-quantification of the endotoxin contamination.

How pH, Temperature, and Time of Incubation Affect False-Positive Responses and Uncertainty of the LAL Gel-Clot Test

The limulus amebocyte lysate (LAL) test is the simplest and most widely used procedure for detection of endotoxin in parenteral drugs. The LAL test demands optimal pH, ionic strength, temperature, and time of incubation. Slight changes in these parameters may increase the frequency of false-positive responses and the estimated uncertainty of the LAL test. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. LAL tests were performed in nominal conditions (37 °C, 60 min, and pH 7) and in different conditions of temperature (36 °C and 38 °C), time of incubation (58 and 62 min), and pH (6 and 8). Slight differences in pH increase the frequency of false-positive responses 5-fold (relative risk 5.0), resulting in an estimated of uncertainty 7.6%. Temperature and time of incubation affect the LAL test less, showing relative risks of 1.5 and 1.0, respectively. Estimated uncertainties in 36 °C or 38 °C temperatures and 58 or 62 min of incubation were found to be 2.0% and 1.0%, respectively. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses. The limulus amebocyte lysate (LAL) gel-clot test is a simple test for detection of endotoxin from Gram-negative bacteria. The test is based on a gel formation when a certain amount of endotoxin is present; it is a pass/fail test. The LAL test requires optimal pH, ionic strength, temperature, and time of incubation. Slight difference in these parameters may increase the frequency of false-positive responses. The aim of this paper is to evaluate how changes in the pH, temperature, and time of incubation affect the occurrence of false-positive responses in the LAL test. We find that slight differences in pH increase the frequency of false-positive responses 5-fold. Temperature and time of incubation affect the LAL test less. Simultaneous differences in these parameters significantly increase the frequency of false-positive responses.

Electrochemical investigation of endotoxin induced limulus amebocyte lysate gel-clot process

Endotoxin can interact with specific receptors on immune cells, leading to inflammatory responses. Thus, it should be strictly controlled in medical devices and pharmaceuticals. The standard endotoxin assay is based on limulus amebocyte lysate (LAL). Herein, we have successfully characterized the electrochemical behavior of endotoxin induced limulus amebocyte lysate gel-clot process for the first time. Screen-printed electrodes with high stability, convenient operation, low cost are produced for the electrochemical investigation. The electrochemical signals of gel-clot process are then applied to endotoxin assay. Compared with other assays, our proposed electrochemical method has faster responses and is more sensitive, anti-jamming and cost-effective, which might have potential use in the clinical applications in the future.

Prevotella bivia as a source of lipopolysaccharide in the vagina

Objectives To compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA). Methods Vaginal washes obtained from patients with ( n = 43) and without ( n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts. Results The median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log 10 CFU/ml, respectively, P < 0.001). Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS). The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV. Conclusion P. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.

Analysis of Endotoxin Residues on Cleaned Implant Materials

Abstract In this study we have investigated the effect of bacteria-containing metal working fluids on the biological residues, which may be found on orthopedic implants. Thus, test coupons and unfinished implants made from commercial pure titanium and titanium aluminum niob were immersed in metal-working fluids containing Pseudomonas pseudoalcaligenes bacteria. Subsequently, these samples were cleaned by means of a multi-tank industrial cleaning scheme, which is commonly used in the medical device industry. The samples were then analyzed with a rapid screening test for bacteria, a Ps. pseudoalcaligenes, specifically, quantitative polymerase chain reaction (PCR) protocol for DNA detection and the Limulus Amebocyte Lysate gel clot test for endotoxins. None of the test pieces showed any biological residues after the cleaning process. There is no increased risk regarding biological residues for metallic implants that were in contact with bacteria containing metal-working fluids if the devices are properly cleaned. A higher risk for biological contamination would more likely arise from any processes after the final cleaning, i.e., from handling, environmental factors, or packaging of the materials.

Airborne Endotoxin and Its Relationship to Pulmonary Function among Workers in an Indian Jute Mill

Air samples from various processing areas of an Indian jute mill were examined for endotoxin. The authors assessed work-related respiratory symptoms and pulmonary function changes in the dust-exposed workers from the different processing areas using a standard questionnaire and spirometry. Endotoxin was estimated in water extract of jute dust from 3 milling areas, and in outside air, by the Limulus amebocyte lysate gel clot technique. The batching, spinning, and weaving areas of the jute mill showed endotoxin levels of 0.22-4.42 microg/m3, 0.04-1.47 microg/m3, and 0.01-0.07 microg/m3, respectively, values similar to those found in Indian cotton mills. Respiratory morbidities among the workers included typical byssinotic symptoms, along with acute changes in postshift forced expiratory volume in 1 s (FEV1.0) (31.8%). Results of this study demonstrated that increased exposure to bacterial endotoxin in airborne dust is related to byssinotic symptoms among Indian jute mill workers. Findings were similar to those reported previously for workers in the cotton, flax, and hemp industries.

False positive endotoxin results in a DC product caused by (1-->3)-beta-D-glucans acquired from a sterilizing cellulose filter.

We prepared autologous DCs to support a Phase I clinical trial of HIV immunization in normal healthy volunteers. Final product-release testing included an assay for endotoxin, performed by the Limulus amebocyte lysate (LAL) gel clot method (Associates of Cape Cod Inc., Falmouth, MA, USA) with a cut-off of 0.06 endotoxin units/mL (EU/mL). In one recent DC preparation, the endotoxin assay was positive (0.48 EU/mL), and the product could not be released for clinical use. There were no overt signs of product contamination or bioburden; we therefore investigated other causes of the abnormal endotoxin results. We filtered all media preparations and the injectable solution for resuspension of the final product using standard laboratory sterile filters prior to use. Of note was the fact that we had used a different type of 0.2 l m filter, made of cellulose nitrate (115 mL Disposable Filterware, Nalgene Company, Rochester, NY, USA), rather than the usual synthetic polyether sulfone (PES) filter (Steriflip, Millipore, Bedford, MA, USA) for this DC preparation, and we suspected that the new filter could be the source of the contamination. We therefore repeated the LAL gel clot assays on the separate ingredients of the DC preparation — USP normal saline, autologous plasma, and the DCs themselves — before and after filtration with the Nalgene filter. Each of the ingredients was negative for endotoxin before filtration, but positive after filtration. We suspected that the ultimate source of the LAL gel clot reactivity in the cellulose nitrate filters could be

Removal of endotoxin from recombinant protein preparations.

To develop an effective method to remove endotoxin from large scale E. coli recombinant protein purifications. Triton X-114 phase separation, affinity chromatography utilizing immobilized polymyxin B or immobilized histidine, were used to remove endotoxin from purified preparations of recombinant CK-BB, CK-MB, CK-MM, myoglobin, and cardiac troponin I. Endotoxin levels were measured by a Limulus Amebocyte Lysate gel-clot assay. The immunoactivity of these protein preparations was determined by BIAcore analysis using a panel of in-house generated monoclonal antibodies and by a Stratus Fluorometric Analyzer. In the case of troponin I, the BIAcore was also utilized to measure troponin C interactions. Phase separation with Triton X-114 was the most effective method in reducing the amount of endotoxin present in the protein preparations compared to either polymyxin B or histidine affinity chromatography. With Triton X-114, the reduction in endotoxin levels was greater than 99% and recovery of the proteins after endotoxin removal was greater than 90%. All three procedures for removing endotoxin had no deleterious effects on the immunoactivity of majority proteins when tested with a panel of monoclonal antibodies. Troponin I also retained its ability to bind to troponin C in the presence of Ca2+. Recombinant CK-BB and CK-MM which were expressed in the soluble fraction of E. coli cell lysates, contained significantly higher endotoxin levels than recombinant CK-MB, myoglobin and cardiac troponin I which were expressed in the form of inclusion bodies. Of the three methods tested, Triton X-114 phase separation was the most effective way of removing endotoxin from recombinant proteins.