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Portelite™ Rapid Fluorimetric Endotoxin Detection Kit

E.coli Endotoxin dose response was measured in a 0.2 mL, thin-wall PCR tube using CytoCite™ with Green fluorescent channel. As low as 0.002 EU/mL of E.coli Endotoxin can be detected with 10 minutes incubation.
E.coli Endotoxin dose response was measured in a 0.2 mL, thin-wall PCR tube using CytoCite™ with Green fluorescent channel. As low as 0.002 EU/mL of E.coli Endotoxin can be detected with 10 minutes incubation.
Ordering information
Price ()
Catalog Number60008
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Lipopolysaccharide (LPS), also known as endotoxin, is the major component of the outer membranes of Gram-negative bacteria. LPS is a potent stimulator of the vertebrate innate immune system and can cause fever, septic shock and eventually death. It is also recognized as a biomarker for the detection of bacterial pathogen invasion, and responsible for the development of inflammatory response and endotoxic shock in extreme cases. Detection of LPS in biological materials, such as protein, peptide or antibody sample, is a critical task for biomanufacturing and bioprocessing. Portelite™ Rapid Fluorimetric Endotoxin Detection Kit uses Endotoxin Green™, a sensitive fluorogenic substrate. Endotoxin Green™ can be hydrolyzed in the presence of endotoxins and the Limulus Amebocyte Lysate (LAL), an extract of blood cells from a horseshoe crab, to generate strong green fluorescence. The endotoxin activity is proportional to the fluorescence intensity resulted from the hydrolysis of Endotoxin Green™. The kit is optimized for Cytocite™ and Qubit™ fluorometers, and can detect a broad range of endotoxin (from 1 EU/ml to 0.002EU/ml) present in the sample.

Platform


CytoCite Fluorometer

Excitation480 nm
Emission510-580 nm
Instrument specification(s)0.2 mL, thin-wall PCR tube

Components


Component A: Endotoxin Green™1 vial
Component B: Endotoxin-Free Water1 bottle (25 mL)
Component C: Limulus Amebocyte Lysate1 vial
Component D: E.coli Endotoxin Standard1 vial (100 EU/mL)
Component E: DMSO1 vial (100 µL)

Example protocol


AT A GLANCE

Protocol summary
  1. Prepare Limulus Amebocyte Lysate (LAL) working solution
  2. Add E.coli Endotoxin Standards and test samples (50 µL)
  3. Add Limulus Amebocyte Lysate working solution (50 µL)
  4. Incubate at 37 °C for 30 minutes
  5. Prepare and add Endotoxin Green™ working solution (100 µL)
  6. Monitor fluorescence with CytoCite™ Fluorometer within 10 minutes 

Important
Thaw all the kit components at room temperature before starting the experiment.
All Materials used in the experiment should be endotoxin-free, such as: disposable tubes or 1.5 mL microcentrifuge tubes, disposable pipette tips, and tubes. The cleanliness of all labware is required to accurately detect levels of endotoxin in a given sample.

PREPARATION OF STOCK SOLUTIONS

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

1. Endotoxin Green™ stock solution
For kit# 60008, add 50 µL of DMSO into one vial of Endotoxin Green™ (Component A) to make Endotoxin Substrate stock solution. For kit# 60009, add 500 µL of DMSO into one vial of Endotoxin Green™ (Component A) to make Endotoxin Substrate stock solution.
Note     Keep from light.


2. Limulus Amebocyte Lysate stock solution
For kit# 60008, add 500 µL Endotoxin-Free Water (Component B) to the vial of Limulus Amebocyte Lysate (Component C) to make 5X  Limulus Amebocyte Lysate (LAL) stock solution. For kit# 60009, add 2.5 mL Endotoxin-Free Water (Component B) to the vial of Limulus Amebocyte Lysate (Component C) to make 5X Limulus Amebocyte Lysate (LAL) stock solution.

PREPARATION OF STANDARD SOLUTION

For convenience, use the Serial Dilution Planner:
https://www.aatbio.com/tools/serial-dilution/


E.coli Endotoxin Standard solution
Add 10 µL of 100 EU/mL E.coli Endotoxin Standard solution to 990 µL of Endotoxin-Free Water (Component B) to generate 1 EU/mL E.coli Endotoxin standard solution. Then take 1 EU/mL E.coli Endotoxin Standard solution, and perform 1:3 serial dilutions in Endotoxin-Free Water (Component B) to get serially diluted E.coli Endotoxin Standards 1 to 0.001 EU/mL.

PREPARATION OF WORKING SOLUTION

1. Endotoxin Green™ working solution
For Kit# 60008, add 50 µL of Endotoxin Green™ stock solution into 5 mL of Endotoxin-Free Water (Component B) to make a total volume of 5.05 mL Endotoxin Green™ working solution. For Kit# 60009, add 500 µL of Endotoxin Green™ stock solution into 50 mL of Endotoxin-Free Water (Component B) to make a total volume of 50.5 mL Endotoxin Green™ working solution.
Note     Each test needs 100 µL, please prepare the amount of Endotoxin Green™ working solution as needed and before use. Please also keep the working solution from light after preparation, and use Endotoxin-Free bottle or tube and store unused stock solutions at -20 °C.


2. Limulus Amebocyte Lysate (LAL) working solution
For kit# 60008, add 500 µL of Limulus Amebocyte Lysate (LAL) stock Solution into 2 mL of Endotoxin-Free Water (Component B) to make a total volume of 2.5 mL Limulus Amebocyte Lysate (LAL) working solution. For kit# 60009, add 2.5 mL of Limulus Amebocyte Lysate (LAL) stock Solution into 10 mL of Endotoxin-Free Water (Component B) to make a total volume of 12.5 mL Limulus Amebocyte Lysate (LAL) working solution.
Note     Each test needs 50 uL, please prepare the amount of LAL working solution as needed and before use. Please also keep the working solution from light after preparation, use Endotoxin-Free bottle or tube, and store unused stock solutions at -20 °C.

SAMPLE EXPERIMENTAL PROTOCOL

  1. Prepare E.coli Endotoxin Standards, blank controls or test samples (50 µL) in a 0.2 mL PCR tube (Cat# CCT100).
  2. Add 50 µL of Limulus Amebocyte Lysate working solution to each tube of E.coli Endotoxin Standard, blank control and test samples.
  3. Mix well and incubate for 30 minutes at 37 °C.
  4. Add 100 µL of Endotoxin Green™ working solution to each tube of Endotoxin Standard, blank control, and test samples to make the total assay volume 200 µL/well.
  5. Insert the samples into CytoCite™ and monitor the fluorescence with green fluorescent channel. Follow the procedure appropriate for CytoCite™ Fluorometer. See the link below for detailed instructions: https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer.
    Note     For best results, read between 2 to 10 minutes after adding the working solution.
    Note     50 µL of 25% acetic acid can be added to stop the reaction. 

Citations


View all 33 citations: Citation Explorer
A Validation Study of the Limulus Amebocyte Lysate Test as an End-Product Endotoxin Test for Polyvalent Horse Snake Antivenom
Authors: Sheraba, N. S., Diab, M. R., Yassin, A. S., Amin, M. A., Zedan, H. H.
Journal: PDA J Pharm Sci Technol (2019): 562-571
Utility Of An Automatic Limulus Amebocyte Lysate Kinetic Turbidimetric Test For Endotoxin Screening Of Dialysate Samples
Authors: Uchida, T., Kaku, Y., Hayasaka, H., Kofuji, M., Momose, N., Miyazawa, H., Ueda, Y., Ito, K., Ookawara, S., Morishita, Y.
Journal: Med Devices (Auckl) (2019): 429-433
Evaluation of a portable test system for assessing endotoxin activity in raw milk
Authors: Suzuki, Y., Suzuki, K., Shimamori, T., Tsuchiya, M., Niehaus, A., Lakritz, J.
Journal: J Vet Med Sci (2016): 49-53
Detection of Endotoxin Contamination of Graphene Based Materials Using the TNF-alpha Expression Test and Guidelines for Endotoxin-Free Graphene Oxide Production
Authors: Mukherjee, S. P., Lozano, N., Kucki, M., Del Rio-Castillo, A. E., Newman, L., Vazquez, E., Kostarelos, K., Wick, P., Fadeel, B.
Journal: PLoS One (2016): e0166816
COMPARISON OF QuantiFERON(R) TB GOLD TEST RESULTS BEFORE AND AFTER ENDOTOXIN CONTAMINATION
Authors: Seto, J., Suzuki, Y., Ahiko, T.
Journal: Kekkaku (2016): 49-52
Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study
Authors: Schaumberger, S., Ladinig, A., Reisinger, N., Ritzmann, M., Schatzmayr, G.
Journal: AMB Express (2014): 1
Comparison of Limulus amebocyte lysate test methods for endotoxin measurement in protein solutions
Authors: Chen, L., Mozier, N.
Journal: J Pharm Biomed Anal (2013): 180-5
Evidence for the detection of non-endotoxin pyrogens by the whole blood monocyte activation test
Authors: Hasiwa, N., Daneshian, M., Bruegger, P., Fennrich, S., Hochadel, A., Hoffmann, S., Rivera-Mariani, F. E., Rockel, C., Schindler, S., Spreitzer, I., Stoppelkamp, S., Vysyaraju, K., Hartung, T.
Journal: ALTEX (2013): 169-208
Contamination of nanoparticles by endotoxin: evaluation of different test methods
Authors: Smulders, S., Kaiser, J. P., Zuin, S., Van L and uyt, K. L., Golanski, L., Vanoirbeek, J., Wick, P., Hoet, P. H.
Journal: Part Fibre Toxicol (2012): 41
Gel clot bacterial endotoxin test of FDG: Indian scenario
Authors: Sharma, S., Mittal, B. R., Vatsa, R., Singh, B.
Journal: Indian J Nucl Med (2011): 149-52