Limulus amebocyte lysate test

Manufactured by Charles River Laboratories

Sourced in United States

Limulus amebocyte lysate (LAL) test is a laboratory analysis method used to detect and quantify the presence of endotoxins, which are lipopolysaccharides derived from the cell walls of Gram-negative bacteria. The test utilizes the unique clotting reaction that occurs when LAL, extracted from the blood cells (amebocytes) of the horseshoe crab, is exposed to endotoxins. This reaction is the basis for the LAL test, which provides a sensitive and accurate means of measuring endotoxin levels in a variety of samples, including pharmaceuticals, medical devices, and other products where the presence of endotoxins must be carefully monitored.

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Lab products found in correlation

Potassium phosphate monobasic, by Merck Group (1 mentions) Pancreatin, by Merck Group (1 mentions) Em11302, by Charles River Laboratories (1 mentions) Polymyxin b sulfate pmb, by AppliChem (1 mentions) Lipopolysaccharide lps from e coli o55 b5, by Merck Group (1 mentions) Sodium hydroxide (naoh), by Merck Group (1 mentions) Amicon ultra 2 centrifugal filter device, by Merck Group (1 mentions)

5 protocols using limulus amebocyte lysate test

Evaluating Endotoxin Removal by Clays

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Lipopolysaccharide (LPS) from E. coli O55:B5 (≥ 500,000 EU/mg) was purchased from Sigma-Aldrich Co. LLC (Vienna, AT). Polymyxin B- sulfate (PMB), an antibiotic, which is known to bind and inactivate endotoxins, was obtained from AppliChem (Darmstadt, D). Two bentonites (Bentonite 1 – main part smektite), one being a natural sodium-bentonite (Bentonite 2 – main part consist of smectite, feldspar and gypsum), and two clays treated with amines (organophilised) were used: a smectite (Organoclay 1 – fully organophilised: whole surface coated with amines) consisting of montmorillonite and an attapulgite (magnesium phylosillicate) consisting of smectite and palygorskite (Organoclay 2 – part of the surface organophilised).
Reagents used for the Limulus amebocyte lysate (LAL) test were obtained from Charles River Laboratories (CRIVER), Inc. Charleston, US: Limulus amebocyte lysate (Endochrome K; Charge: C4452E), Endotoxin-free (< 0.005 EU/mL) LAL reagent water (LRW; Charge: 99732088) and Endosafe control Standard Endotoxin from E. coli O55:B5 1.000 EU/mL (Charge: EX 14392 and EM11302 – RSE/CSE ratio 10 EU/ng; EX01022 – RSE/CSE ratio 12 EU/ng; EM11302 – RSE/CSE ratio 7 EU/ng). Potassium phosphate monobasic, sodium hydroxide and pancreatin for the preparation of the artificial intestinal fluid were obtained from Merck KGaA (Darmstadt, D).

Schaumberger S., Ladinig A., Reisinger N., Ritzmann M, & Schatzmayr G. (2014). Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study. AMB Express, 4, 1.

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Quantification of PMB and LPS from Pseudomonas and Escherichia coli

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PMB and endotoxins (LPS) from Pseudomonas aeruginosa and Escherichia coli were purchased from Sigma-Aldrich (Vienna, Austria). The PMB analyses were conducted using the Colistin & Polymyxin ELISA kit from Kwinbon Biotechnology Co. LTD (Beijing, China). The analyses of endotoxins were performed in pyrogen-free tubes and the kinetic chromogenic Limulus amebocyte lysate (LAL) test from Charles River Laboratories (Wilmington, MA, USA). Tubes (Vacuettes) for blood donation were obtained from Greiner (Kremsmünster, Austria). The Amicon Ultra-2 centrifugal filter devices with molecular cut-offs of 10, 30, 50 and 100 kDa were from Merck (Darmstadt, Germany). The ALBplus reagent set for albumin quantification and the TP set for measurement of total protein level was purchased from Roche (Mannheim, Germany).

Harm S., Gabor F, & Hartmann J. (2016). Low-dose polymyxin: an option for therapy of Gram-negative sepsis. Innate Immunity, 22(4), 274-283.

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Quantifying LPS Neutralization in Serum

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The LPS neutralization study in serum was conducted using a chromogenic Limulus Amebocyte Lysate (LAL) test (Charles River Laboratories, MA, USA). The LAL test is the most established method for quantifying LPS. This method uses amebocyte lysate from Limulus polyphemus, wherein the presence of LPS triggers a defense mechanism that induces coagulation. The LAL test is used successfully for detection of LPS in a wide variety of industrial, pharmaceutical, and research applications, such as water supplies, parenteral fluids, drugs for intravenous administration, and certain biologic fluids (e.g., cerebrospinal fluid). Therefore, serum, which was obtained from freshly drawn blood, was spiked with 500 ng/mL AMPs, PMB or LL-37 before adding 5 ng/mL LPS from E. coli or P. aeruginosa. As positive and negative controls, native serum with and without LPS was used, respectively. Endotoxin activity was measured using the LAL assay according to the manufacturer’s specifications.

Harm S., Lohner K., Fichtinger U., Schildböck C., Zottl J, & Hartmann J. (2019). Blood Compatibility—An Important but Often Forgotten Aspect of the Characterization of Antimicrobial Peptides for Clinical Application. International Journal of Molecular Sciences, 20(21), 5426.

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Triton X-114 Endotoxin Removal

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For LPS removal, Triton X-114 (Sigma-Aldrich Seelze, Germany) was added to the purified protein solution to a final concentration of 1%. After shaking at +4 °C for 20 minutes, phase separation was done by shaking at 25 °C for 10 minutes. Samples were then centrifuged for 10 minutes at 25 °C, 17000 × g. The upper (Triton X-114-poor) phase was collected, and Triton X-114 was added again to the same final concentration. Separation was repeated and the supernatant was dialyzed extensively against PBS for 3 days with changing the buffer every 12 h. The final LPS-depleted protein solution was concentrated, and endotoxin content was measured by limulus amebocyte lysate test (Charles River, Freiburg, Germany) according to the manufacturer’s instructions.

Akhras S., Toda M., Boller K., Himmelsbach K., Elgner F., Biehl M., Scheurer S., Gratz M., Vieths S, & Hildt E. (2017). Cell-permeable capsids as universal antigen carrier for the induction of an antigen-specific CD8+ T-cell response. Scientific Reports, 7, 9630.

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Characterization of Mesoporous Silica Rods

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All MSR materials were analyzed before in vitro T-cell activation. Thermogravimetric analysis (TGA) was conducted to confirm removal of contaminants and the individual MSRs were processed for sizing via bright field microscopy (EVOS FL Cell Imaging System) and scanning electron microscopy (SEM) to determine rod dimensions and ultrastructure. Nitrogen physisorption (3Flex) was performed to determine the pore size, volume, and surface area of the MSRs. These analyses are summarized in Supplementary Table 2. MSRs were also evaluated for their ability to coat liposomes and release IL-2, relevant biological proxies to confirm consistent physical structure between batches. MSRs were further confirmed to be endotoxin free via the Limulus Amebocyte Lysate test (Charles River).

Zhang D.K., Adu-Berchie K., Iyer S., Liu Y., Cieri N., Brockman J.M., Neuberg D., Wu C.J, & Mooney D.J. (2023). Enhancing CAR-T cell functionality in a patient-specific manner. Nature Communications, 14, 506.

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