Limulus amebocyte lysate test

Manufactured by Associates of Cape Cod

Sourced in United States

Limulus amebocyte lysate (LAL) test is a laboratory assay used to detect and quantify bacterial endotoxin or lipopolysaccharide (LPS). It utilizes the clotting reaction of the amebocyte cells from the horseshoe crab (Limulus polyphemus) to indicate the presence of endotoxin in a sample.

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Lab products found in correlation

Microplate reader, by Thermo Fisher Scientific (2 mentions) Affinitypak detoxi gel endotoxin removing gel, by Thermo Fisher Scientific (1 mentions) Isopropylthio β galactoside iptg, by Merck Group (1 mentions) Bicinchoninic acid protein assay kit, by Beyotime (1 mentions) Fitc dextran, by Thermo Fisher Scientific (1 mentions) Fitc dextran, by Merck Group (1 mentions) Safe lock microcentrifuge tube, by Eppendorf (1 mentions) Heparanase assay kit, by AMS Biotechnology (1 mentions) Polymyxin b, by Merck Group (1 mentions) Hmec 1, by American Type Culture Collection (1 mentions) Endofree plasmid maxi, by Qiagen (1 mentions)

8 protocols using limulus amebocyte lysate test

Monocyte and Platelet Incubation with Anti-β2-GPI IgG

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For in vitro studies, monocytes were cultured at 37°C in a humified atmosphere of 5% CO₂ with serum-free RPMI 1640, containing 2 mM L-glutamine, 100 units/ml of penicillin, 100 mg/ml of streptomycin, and 250 pg/ml of Fungizone. Platelets were resuspended in Tyrode's buffer, containing BSA (3 mg/ml).
Monocytes (2 × 10⁶/ml) or platelets (300 × 10⁶/ml) were incubated at 37°C for 4 h with human affinity-purified anti-β₂-GPI IgG (200 μg/ml), according to the method of Raschi et al. (25 (link)) with normal human serum IgG (200 μg/ml), or with LPS (100 ng/ml). All materials contained < 0.00025 ng of endotoxin/μg of protein, as determined by the Limulus amebocyte lysate test (Associates of Cape Cod, Falmouth, MA).

Manganelli V., Truglia S., Capozzi A., Alessandri C., Riitano G., Spinelli F.R., Ceccarelli F., Mancuso S., Garofalo T., Longo A., Valesini G., Sorice M., Conti F, & Misasi R. (2019). Alarmin HMGB1 and Soluble RAGE as New Tools to Evaluate the Risk Stratification in Patients With the Antiphospholipid Syndrome. Frontiers in Immunology, 10, 460.

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Evaluating Household Exposure to Endotoxin

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Parents were administered a questionnaire gathering information on parental and child health in the previous year and environmental exposures. Environmental tobacco smoke (ETS) was categorically defined as parental report of at least one current smoker residing in the household. House dust samples were collected from a 2 m² area of floor surface from the infant’s primary living area [22 (link)]. Endotoxin concentrations were determined by the limulus amebocyte lysate test (Associates of Cape Cod Inc, Falmouth, MA, USA) in all house dust samples according to methods described by Milton and colleagues [23 (link)].

Cheng G., Smith A.M., Levin L., Epstein T., Ryan P.H., LeMasters G.K., Hershey G.K., Reponen T., Villareal M., Lockey J, & Bernstein D.I. (2014). Duration of day care attendance during infancy predicts asthma at the age of seven: the Cincinnati Childhood Allergy and Air Pollution Study. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology, 44(10), 1274-1281.

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Recombinant Production and Purification of Sj16

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Recombinant Sj16 and GST were produced and purified as previously described 32 (link). Briefly, the indicated plasmid (pGEX-4T-1/Sj16) was constructed and transformed into E. coli BL21 (DE3). rSj16 was expressed as a GST-Sj16 fusion protein via incubation with 1 mM isopropylthio-β-galactoside (IPTG, Sigma, USA) at 37°C. The GST-Sj16 fusion protein was purified using a GSTrap™ FFresin column (Amersham Pharmacia, USA) before being subjected to thrombin enzyme (Amersham, USA) digestion, resulting in the production of rSj16. GST was purified from the column by GST elution buffer and collected, after which the purified rSj16 and GST were excised from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and identified by mass spectrometry and treated with AffinityPak™ Detoxi-Gel™ Endotoxin Removing Gel (Thermo, USA) to eliminate the endotoxin, which was detected using the Limulus amebocyte lysate test (sensitivity 0.25 EU/ml, Associates of Cape Cod, USA). The normal concentration of endotoxin in a 1 mg/ml gel was 0.01 EU/kg and has been found to be as high as 0.2 EU/kg, according to the endotoxin normative standard in the “American FDA Final Product Examination Guide”. The concentrations of rSj16 and GST were determined by Bicinchoninic Acid Protein Assay Kit (Beyotime Biotechnology, China).

Wang L., Xie H., Xu L., Liao Q., Wan S., Yu Z., Lin D., Zhang B., Lv Z., Wu Z, & Sun X. (2017). rSj16 Protects against DSS-Induced Colitis by Inhibiting the PPAR-α Signaling Pathway. Theranostics, 7(14), 3446-3460.

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Gut Permeability Assessment via FITC-Dextran

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Fluorescein isothiocyanate (FITC)-dextran, a gut non-absorbable molecule, was orally administered to determine gut permeability as previously published [65 (link)]. In brief, FITC-dextran (molecular weight 4.4 kDa; Sigma-Aldrich) was administered at 25 mg/mL in 0.25 mL PBS at 3 h before blood collection. Serum FITC-dextran was measured by fluorospectrometry (microplate reader; Thermo Scientific, Wilmington, DE, USA). In addition, serum endotoxin (LPS) was measured as another gut-leakage parameter using the Limulus Amebocyte lysate test (Associates of Cape Cod, East Falmouth, MA, USA) and values of LPS < 0.01 EU/mL were recorded as 0 due to the limitation of the standard curve.

Udompornpitak K., Bhunyakarnjanarat T., Charoensappakit A., Dang C.P., Saisorn W, & Leelahavanichkul A. (2021). Lipopolysaccharide-Enhanced Responses against Aryl Hydrocarbon Receptor in FcgRIIb-Deficient Macrophages, a Profound Impact of an Environmental Toxin on a Lupus-Like Mouse Model. International Journal of Molecular Sciences, 22(8), 4199.

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Comprehensive Biomarker Analysis of Systemic Insult

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Serum endotoxin (LPS) was measured using the Limulus Amebocyte lysate test (Associates of Cape Cod, East Falmouth, MA, USA) and values of LPS < 0.01 EU/mL were recorded as 0 due to the limitation of the standard curve. Kidney injury (serum creatinine and blood urea nitrogen) and liver damage (alanine transaminase—ALT) were determined by QuantiChrom Creatinine-Assay (DICT-500), Urea-assay (DIUR-500) and EnzyChrom ALT assay (EALT-100) (BioAssay, Hayward, CA, USA). Serum cytokines and cell-free DNA (cfDNA) were measured by enzyme-linked immunosorbent assay (ELISA) (Invitrogen, San Diego, CA, USA) and Quanti PicoGreen assay (Sigma-Aldrich), respectively. For peripheral blood leukocytes, blood was mixed with 3% volume by volume (v/v) of acetic acid for red blood cell lysis in a ratio of blood and acetic acid at 1:20 by volume before counting with hemocytometer. In addition, Wright-stained blood smear was determined for the percentage of neutrophils and lymphocytes. The total number of these cells was calculated by the total count from hemocytometer multiplied by the percentage of cells from the Wright-stained slide.

Visitchanakun P., Kaewduangduen W., Chareonsappakit A., Susantitaphong P., Pisitkun P., Ritprajak P., Townamchai N, & Leelahavanichkul A. (2021). Interference on Cytosolic DNA Activation Attenuates Sepsis Severity: Experiments on Cyclic GMP–AMP Synthase (cGAS) Deficient Mice. International Journal of Molecular Sciences, 22(21), 11450.

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Biomarker Analysis of Plasma Samples

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Laboratory workup included the determination of plasma brain natriuretic peptide (BNP), creatinine, C‐reactive protein, LBP, IL‐6 (Central Laboratory unit of University Hospital Halle), IL‐1β, and TNF‐α (amedes, Halle/Leipzig GmbH, Halle, Germany). The determination of endotoxin plasma levels was carried out by the accredited laboratory Dr. Michael Lohmeyer GmbH, Mendelstr. 11, Münster, Germany, using a commercial Limulus Amebocyte Lysate Test (turbidimetric method, Pyrotell‐T®, Associates of Cape Cod, Inc.; reagents by Haemochrom, Essen, Germany). Plasma samples (0.5 mL) were stored in 1.5 mL Eppendorf® Safe‐Lock microcentrifuge tubes at −70°C until analysis was performed.

Linhart C., Ulrich C., Greinert D., Dambeck S., Wienke A., Girndt M, & Pliquett R.U. (2018). Systemic inflammation in acute cardiorenal syndrome: an observational pilot study. ESC Heart Failure, 5(5), 920-930.

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In Vitro Evaluation of Anti-β2-GPI Antibody Effects

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For in vitro studies, HUVECs (5 × 10⁵/mL), HMEC‐1 (American Type Culture Collection, ATCC; 5 × 10⁵/mL, see supporting information) and human platelets (3 × 10⁸/mL) were seeded into 6‐well cell culture and incubated at 37°C, for different incubation times, with affinity‐purified or polyclonal anti‐β2‐GPI antibodies (200 μg/ml), normal human serum IgG (200 μg/ml), or lipopolysaccharide (LPS) (100 ng/ml), according to the methods previously described.⁷ (link), ²⁴ (link) To exclude the possibility of LPS contamination, samples were stimulated in the presence or absence of anti‐β2GPI antibody and then pretreated with polymyxin B (10 μg/ml; Sigma‐Aldrich). In parallel experiments, HUVECs, as well as human platelets, were pretreated with selective heparanase inhibitor RDS3337 (320 nM) for 1 h before treatments. Virtually no heparanase activity was detected in HUVECs and platelets pre‐incubated with RDS3337, as detected by Heparanase Assay kit (Amsbio). On the contrary, affinity‐purified as well as LPS induced a significant increase of heparanase activity and release (Figure S1 in supporting information).
All materials contained <0.00025 ng of endotoxin/μg of protein, as determined by the Limulus amebocyte lysate test (Associates of Cape Cod).

Capozzi A., Riitano G., Recalchi S., Manganelli V., Costi R., Saccoliti F., Pulcinelli F., Garofalo T., Misasi R., Longo A., Di Santo R, & Sorice M. (2021). Effect of heparanase inhibitor on tissue factor overexpression in platelets and endothelial cells induced by anti‐β2‐GPI antibodies. Journal of Thrombosis and Haemostasis, 19(9), 2302-2313.

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Plasmid-based DNA Vaccination Protocol

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To generate plasmids for DNA vaccination, a previously described plasmid expression vector designated as pT.neo was used [26] (link). The antigenic peptide sequences were inserted at the 3′ EcoRI/XbaI flanking site of the T helper epitope of pT.neo. For co-immunization studies, a previously described plasmid encoding for murine interferon-γ (pmIFN-γ) was used [27] (link). The plasmids were produced by transforming DH5a Escherichia coli cells and purified, after sequence analysis, using EndoFree Plasmid Maxi or Giga kits (Qiagen). Each lot of plasmid DNA had a A₂₆₀/A₂₈₀ ratio ≥1.8, as determined by UV spectrophotometry, endotoxin content ≤0.1 EU/µg DNA, as determined by Limulus Amebocyte Lysate test (Associates of Cape Cod) and a predominantly supercoiled form.
For comparison, two selected peptides were conjugated to keyhole limpet haemocyanin (KLH) to increase their antigenicity and immunogenicity.

Polonelli L., Beninati C., Teti G., Felici F., Ciociola T., Giovati L., Sperindè M., Passo C.L., Pernice I., Domina M., Arigò M., Papasergi S., Mancuso G., Conti S, & Magliani W. (2014). Yeast Killer Toxin-Like Candidacidal Ab6 Antibodies Elicited through the Manipulation of the Idiotypic Cascade. PLoS ONE, 9(8), e105727.

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