Amplite® Fluorimetric Endotoxin Detection Kit

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Telephone	1-800-990-8053
Fax	1-800-609-2943
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H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
UNSPSC	12171501

Overview SDS Protocol

Lipopolysaccharide (LPS), also known as endotoxin, is the major component of the outer membrane of Gram-negative bacteria. LPS is a potent stimulator of the vertebrate innate immune system and can cause fever, septic shock and eventually death. LPS is also recognized as a biomarker for the detection of bacterial pathogen invasion, and is 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 in biological manufacturing and processing. Amplite® Fluorimetric Endotoxin Detection Kit uses Endotoxin Green™, a sensitive fluorogenic substrate. Endotoxin Green™ is hydrolyzed in the presence of endotoxins and the Limulus Amebocyte Lysate (LAL), an extract of blood cells from a horseshoe crab. The hydrolyzed product of Endotoxin Green™ generates strong green fluorescence. The endotoxin activity is proportional to the fluorescence intensity resulted from the hydrolysis of Endotoxin Green™. Amplite® Fluorimetric Endotoxin Detection Kit can detect a broad range of endotoxin (from 1 EU/ml to 0.001 EU/ml). It is very sensitive and can detect as low as 0.001 EU/mL of endotoxin within 30 min.

Platform

Fluorescence microplate reader

Excitation	490 nm
Emission	525 nm
Cutoff	515 nm
Recommended plate	Solid black
Instrument specification(s)	Top read mode

Components

Example protocol

AT A GLANCE

Protocol summary

Prepare Endotoxin Green™ working solution
Add E.coli Endotoxin Standards and test samples (25 µL)
Add Limulus Amebocyte Lystate solution (25 µL)
Incubate at 37 °C for 30 minutes
Add Endotoxin Green™ working solution (50 µL)
Read fluorescence intensity at Ex/Em=490/525 nm 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 disposable 96-well microplates or plate strips. 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. 100 X Endotoxin Green™ stock solution

Add 50 µL of DMSO into the vial of Endotoxin Green™ (Component A) to make 100X Endotoxin Green™ stock solution.
Note Keep from light.

2. 5X Limulus Amebocyte Lysate solution

Add 500 µL of Endotoxin-Free Water (Component B) into the vial of Limulus Amebocyte Lysate (Component C) to make 5X Limulus Amebocyte Lysate 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 (ES1). Then take 1 EU/mL E.coli Endotoxin Standard solution (ES1) and perform 1:3 serial dilutions in Endotoxin-Free Water (Component B) to get serially diluted E.coli Endotoxin Standards (ES2 - ES7).

PREPARATION OF WORKING SOLUTION

1. Endotoxin Green™ working solution

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.
Note Prepare the amount of Endotoxin Green™ working solution as needed. Keep the working solution from light.

2. Limulus Amebocyte Lysate (LAL) working solution

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.
Note Prepare the amount of LAL working solution as needed and before use. Using the Endotoxin-Free bottle or tube.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1.Layout of E.coli Endotoxin Standards and test samples in a solid black 96-well microplate. ES=E.coli Endotoxin standards (ES1-ES7, 1.00 to 0.001 EU/mL); BL=Blank Control; TS=Test Samples

BL	BL	TS	TS
ES1	ES1	...	...
ES2	ES2	...	...
ES3	ES3
ES4	ES4
ES5	ES5
ES6	ES6
ES7	ES7

Table 2.Reagent composition for each well.

Well	Volume	Reagent
ES1-ES7	25 µL	Serial dilutions (1.00-0.001 EU/mL)
BL	25 µL	Endotoxin-Free Water
TS	25 µL	Test Samples

Prepare E.coli Endotoxin Standards (ES), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 12.5 µL of reagent per well instead of 25 µL.
Add 25 µL of 2X Limulus Amebocyte Lysate solution to each well of E.coli Endotoxin Standard, blank control and test samples.
Mix well and incubate for 30 minutes at 37 °C.
Add 50 µL of Endotoxin Green™ working solution to each well of E.coli Endotoxin Standard, blank control, and test samples to make the total assay volume 100 µL/well. For a 384-well plate, add 25 µL of Amplite™ Endotoxin Green working solution into each well instead, for a total volume of 50 µL/well.
Monitor the fluorescence intensity at Ex/Em=490/525 nm, cutoff 515 nm.
Note For best results, read between 2 to 10 minutes after adding the working solution.
Note 25 µL of 25% acetic acid can be added to stop the reaction.

Images

Figure 1. E.coli endotoxin dose response was measured in a black/solid bottom 96-well plate using a Gemini microplate reader (Molecular Devices) at Ex/Em=490/525 nm, cutoff=515 nm. As low as 0.001 EU/mL of E.coli Endotoxin can be detected after incubation (n=3).

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