Lal qcl 1000 assay

Manufactured by Lonza

Sourced in Switzerland, United States

The LAL QCL-1000 assay is a quantitative endotoxin detection system used to measure the presence and concentration of bacterial endotoxins in pharmaceutical and medical device products. The assay utilizes the Limulus Amebocyte Lysate (LAL) reaction to detect and quantify endotoxin levels. The system provides a reliable and accurate method for ensuring product safety and quality control.

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Lab products found in correlation

Human quantikine kits, by R&D Systems (1 mentions) Hk315, by Hycult Biotech (1 mentions) Hitrap q hp, by GE Healthcare (1 mentions) 293f cells, by Thermo Fisher Scientific (1 mentions) Biofrac fraction collector, by Bio-Rad (1 mentions) Ngc chromatography system, by Bio-Rad (1 mentions) Ova grade 5, by Merck Group (1 mentions) Isoflo, by Abbott (1 mentions) Quantikine enzyme linked immunosorbent assay system, by R&D Systems (1 mentions) Anti his antibody, by Thermo Fisher Scientific (1 mentions)

15 protocols using lal qcl 1000 assay

Quantification of Inflammatory Markers in Liver Disease

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To measure BT-related markers and proinflammatory cytokines, peripheral blood samples were collected on the same date of pre- and post-treatment HVPG measurements. Blood samples were separated by centrifugation at 1,500×g for 15 minutes within 30 minutes of collection. Samples were stored at −80°C until the analysis.
To determine LPS, serum samples were diluted 1:10 with pyrogen-free water and incubated for 10 minutes at 75°C to remove serum inhibitors. The concentration of LPS in serum was analyzed using the Limulus Amebocyte Lysate (LAL) assay, a quantitative chromogenic test for detecting endotoxins (QCL-1000 LAL assay; Lonza, Walkersville, MD, USA). The lower detection limit for LPS was 0.1 EU/mL. ELISA assays were used to quantitatively measure the serum concentration of LBP (HK315; Hycult Biotech, Uden, The Netherlands), IL-6 (Human Quantikine kits; R&D Systems, Minneapolis, MN, USA), and TNF-α (Human Quantikine kits) according to the manufacturers’ instructions. The absorbance at the 450-nm light wave was measured in each well with a microplate reader (BioTek ELX; BioTek, Shoreline, WA, USA). The lower limit of detection for LBP was 4.4 ng/mL, IL-6 was 0.70 pg/mL, and TNF-α was 0.5 pg/mL. All measurements were performed in duplicate and the mean value is presented.

Lim Y.L., Kim M.Y., Jang Y.O., Baik S.K, & Kwon S.O. (2017). Rifaximin and Propranolol Combination Therapy Is More Effective than Propranolol Monotherapy for the Reduction of Portal Pressure: An Open Randomized Controlled Pilot Study. Gut and Liver, 11(5), 702-710.

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Anion Exchange Chromatography Purification

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The protein of interest was exchanged into a 20 mM buffer suitable for anion exchange chromatography at a pH one unit below its isoelectric point, with 150 mM NaCl in addition. The isoelectric points were calculated using Geneious software (Biomatters, Auckland, New Zealand). The flow-through was collected after repeated passages through a 5 mL HiTrap Q HP anion exchange column (GE Healthcare, Pittsburgh, PA), and endotoxin levels were measured using the QCL-1000 LAL assay following the manufacturer’s instructions (Lonza, Basel, Switzerland).

Yang N.J., Liu D.V., Sklaviadis D., Gui D.Y., Vander Heiden M.G, & Wittrup K.D. (2015). Antibody-Mediated Neutralization of Perfringolysin O for Intracellular Protein Delivery. Molecular pharmaceutics, 12(6), 1992-2000.

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Purification and Characterization of Salivary Proteins from Lu. intermedia

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Eleven Lu. intermedia cDNA coding for secreted proteins (LinB-8, LinB-13, LinB-14, LinB-15, LinB-17, LinB-21, LinB-26, LinB-28, LinB-37, LinB-38, LinB-44) (Table 1)^{17 (link)} were amplified by PCR, cloned into VR2010-TOPO and sequenced as described^{45 (link)}. Recombinant proteins were produced by transfection of 293-F cells (Invitrogen) with VR2010-TOPO plasmids coding for different Lu. intermedia salivary proteins by the Protein Expression Laboratory at the Frederick National Laboratory for Cancer Research (Frederick, MD). High-performance liquid chromatography purification of His-tagged Lu. intermedia salivary proteins was performed as described^{6 (link)} using an NGC Chromatography System (BioRad). Eluted proteins were detected at 280 nm and collected every minute on a 96-well microtiter plate using a BioFrac Fraction Collector (BioRad). Recombinant proteins were tested for endotoxin using a QCL-1000 LAL assay (Lonza). Endotoxin-free recombinant proteins were used in all assays. Aliquots of eluted proteins were checked by SDS-PAGE developed by silver staining. Edman sequencing of the N-terminus region of the purified recombinant proteins was performed at the Protein Chemistry Section, Research Technology Branch, NIAID, NIH (Rockville, MD). Proteins were stored at −70 °C until use.

Carvalho A.M., Fukutani K.F., Sharma R., Curvelo R.P., Miranda J.C., Barral A., Carvalho E.M., Valenzuela J.G., Oliveira F, & de Oliveira C.I. (2017). Seroconversion to Lutzomyia intermedia LinB-13 as a biomarker for developing cutaneous leishmaniasis. Scientific Reports, 7, 3149.

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Murine Model of Allergic Sensitization

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Mice were anesthetized with isoflurane (IsoFlo; Abbott Laboratories,
Chicago, Ill) and their backs shaved with an electric razor 1 or 2 days before
first allergen exposure. A total of 250 μL of sterile saline solution,
OVA (albumin from chicken egg white, OVA grade V, Sigma-Aldrich, St Louis, Mo),
ASP (XPM110D3A4; Greer, Lenoir, NC), or both at a concentration of 1 mg/mL was
applied to a 2 × 2 cm patch of sterile gauze. The patch was secured with
TegaDerm (3M, Maplewood, Minn), and the mouse was wrapped with a Band-Aid and
waterproof tape. After 6 days, the patch was removed; 24 hours later, a new
patch was applied, for a total of 3 patches over a 3-week period, as shown in
Fig 1, A. Endotoxin levels in OVA and
ASP were assessed using the QCL-1000 LAL assay (Lonza, Basel, Switzerland): 1
mg/mL of OVA grade V contains about 250 EU/mL of endotoxin (50 EU/mL per patch),
and 1 mg/mL of ASP contains between 0.04 EU/mL (lot 357280) and 0.14 EU/mL (lot
194776).

Minor S.Y., Banerjee A, & Gotschlich E.C. (2000). Effect of α-Oligosaccharide Phenotype of Neisseria gonorrhoeae Strain MS11 on Invasion of Chang Conjunctival, HEC-1-B Endometrial, and ME-180 Cervical Cells. Infection and Immunity, 68(12), 6526-6534.

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Inflammatory Cytokine Profiling and LPS Quantification

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IL-23, IL-17A, IL-8, TNF-α, GM-CSF, and IFN-γ were measured by ELISA kits (R&D Systems). Arginase I levels were also determined by an ELISA kit (BioVendor). Tissue LPS levels were analyzed in duplicate wells in 96-well plates according to the manufacturer’s instructions with Limulus Amoebocyte Lysate (LAL) assay QCL-1000 (Lonza, ValaisSwitzerland). The LPS levels were normalized back to the weight of the tissue samples used.

Wu P., Wu D., Ni C., Ye J., Chen W., Hu G., Wang Z., Wang C., Zhang Z., Xia W., Chen Z., Wang K., Zhang T., Xu J., Han Y., Zhang T., Wu X., Wang J., Gong W., Zheng S., Qiu F., Yan J, & Huang J. (2014). γδT17 Cells Promote the Accumulation and Expansion of Myeloid-Derived Suppressor Cells in Human Colorectal Cancer. Immunity, 40(5), 785-800.

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LPS and LBP Quantification in Serum

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Blood was collected via cardiac puncture. LPS and LBP detection in the serum were based on a previously performed method (20 (link)). Circulating endotoxin (LPS) was analyzed using a Limulus Amoebocyte Lysate (LAL) assay QCL-1000 (Lonza AG, Valais Switzerland) in duplicate in 96-well plates according to the manufacturer's instructions; lipopolysaccharide-binding protein (LBP) levels were detected using the LBP Elisa kit (Abnova, Taipei, Taiwan). The lower limit of detection for each assay is 0.1 EU/ml for LPS and 5 ng/ml for LBP.

Yang Y., Qu C., Liang S., Wang G., Han H., Chen N., Wang X., Luo Z., Zhong C., Chen Y., Li L, & Wu W. (2017). Estrogen inhibits the overgrowth of Escherichia coli in the rat intestine under simulated microgravity. Molecular Medicine Reports, 17(2), 2313-2320.

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Quantifying Endotoxin and sCD163 Levels

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Plasma endotoxin (LPS) levels: Samples were diluted with LAL reagent in a ratio of 1:3 and heated at a temperature of 65 °C for half an hour. All samples were analyzed in replica according to the instructions, using the Limulus Amoebocyte Lysate (LAL) assay QCL-1000 (Lonza, Valais Switzerland).
Soluble CD163 (sCD163): Blood samples were collected in a 10-mL EDTA-coated tube, centrifuged and serum was then stored at − 80 °C. The samples were thawed and serum sCD163 was analyzed using the Quantikine enzyme-linked immunosorbent assay system (R&D Systems, Minneapolis, MN). The mean assay coefficient of variance was 3.3% for sCD163.

Hegazy M.A., Mogawer S.M., Alnaggar A.R., Ghoniem O.A, & Abdel Samie R.M. (2020). Serum LPS and CD163 Biomarkers Confirming the Role of Gut Dysbiosis in Overweight Patients with NASH. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 13, 3861-3872.

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Quantifying Plasma LPS and CRP

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CB samples were centrifuged twice to separate plasma, which was stored at −80°C for batch analysis. Plasma was diluted to 10% or 20% with endotoxin free water and then heated to 85°C for 15 minutes to denature plasma proteins. Plasma levels of LPS were measured using a commercially available kit (Limulus Amebocyte Lysate [LAL] assay QCL-1000, Lonza, Walkersville, MD) according to the manufacturer's protocol. The lower level of detection for the assay was 0.7 pg/ml. Plasma levels of CRP were measured using a commercially available sandwich enzyme immunoassay kit and following manufacturer's protocol (Quantikine ELISA DCRP00, R&D, Minneapolis, MN). The minimum level of detection was 0.01 ng/ml.

Luciano A.A., Arbona-Ramirez I.M., Ruiz R., Llorens-Bonilla B.J., Martinez-Lopez D.G., Funderburg N, & Dorsey M.J. (2014). Alterations in Regulatory T Cell Subpopulations Seen in Preterm Infants. PLoS ONE, 9(5), e95867.

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Endotoxin Analysis via LAL Assay

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Samples were diluted 1:3 with LAL reagent water and heat inactivated for 30min at 65°C. All samples were analyzed in duplicate according to the manufacturer’s instructions, using the Limulus Amoebocyte Lysate (LAL) assay QCL-1000 (Lonza, Valais Switzerland).

du Plessis J., Korf H., van Pelt J., Windmolders P., Vander Elst I., Verrijken A., Hubens G., Van Gaal L., Cassiman D., Nevens F., Francque S, & van der Merwe S. (2016). Pro-Inflammatory Cytokines but Not Endotoxin-Related Parameters Associate with Disease Severity in Patients with NAFLD. PLoS ONE, 11(12), e0166048.

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Production and Purification of Recombinant Proteins

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Recombinant (fusion) proteins were produced as previously described [13] (link). In short, gene amplified PCR products were cloned by Gateway Technology (Invitrogen, San Diego, CA, USA) in a bacterial expression vector containing an N-terminal hexa-histidine (His) tag. Generated vectors were sequenced to confirm correct insertion of the product. Recombinant proteins were overexpressed in Escherichia coli strain BL21 (DE3) and further purified. The size and purity of the proteins were analyzed by gel electrophoresis, using Coomassie brilliant blue and Western blotting using an anti-His antibody (Invitrogen, Carlsbad, CA, USA). Endotoxin contents were below 50 EU (endotoxin unit) per mg of recombinant protein as tested using a Limulus Amebocyte Lysate (LAL) QCL-1000 assay (LonzaInc., Basel, Switzerland). Recombinant proteins were tested to exclude protein non-specific T-cell stimulation and cellular toxicity in IFN-γ release assays using PBMC of in vitro PPD-negative, healthy Dutch donors recruited at the Blood Bank Sanquin, Leiden, The Netherlands [14] (link), [15] (link). None of these controls had experienced any known prior contact with TB patients.

Commandeur S., Coppola M., Dijkman K., Friggen A.H., van Meijgaarden K.E., van den Eeden S.J., Wilson L., van der Ploeg-van Schip J.J., Franken K.L., Geluk A, & Ottenhoff T.H. (2014). Clonal Analysis of the T-Cell Response to In Vivo Expressed Mycobacterium tuberculosis Protein Rv2034, Using a CD154 Expression Based T-Cell Cloning Method. PLoS ONE, 9(6), e99203.

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