ToxinSensor Chromogenic LAL Endotoxin Assay Kit

Catalog No. ABIN491527

Product Details

Reactivity: Human, Various Species

Detection Range: 0.005-1 U/mL

Minimum Detection Limit: 0.005 U/mL

Application: Cytotoxicity Test (CyTox)

Brand: ToxinSensor™

Characteristics: ToxinSensor ^TM Chromogenic LAL Endotoxin Assay Kit is designed to be used as a quantitative In Vitro end-point endotoxin test for human and animal parenteral drugs, biological products, and medical devices. This method utilizes a modified Limulus Amebocyte Lysate and a synthetic color producing substrate to detect endotoxin chromogenically. The kit has a minimum endotoxin detection limit of 0.005 EU/mL and a measurable concentration range of 0.005 to 1 EU/mL. In addition, any sample with color (e.g. bacterial/cell culture medium, serum or blood etc.) cannot be assayed using this kit.

Components: LAL Reagent Water, 50 mL/bottle (1 bottle)
Limulus Amebocyte Lysate (LAL) (1 Vial)
E. coli Endotoxin Standard (1 Vial)
Chromogenic Substrate (1 Vial)
Buffer S for Color-stabilizer #1 (10 mL)
Color-stabilizer #1 (1 Vial)
Color-stabilizer #2 (1 Vial)
Color-stabilizer #3 (1 Vial)
Endotoxin-free tubes (25)
Endotoxin-free Tips, 200 µL (1 box (96 tips))
Endotoxin-free Tips, 1000 µL (2 bags (12 tips))
Incubation Rack (1)

Material not included: 1. Sodium hydroxide, 0.1 N, dissolved in LAL Reagent Water, for pH adjustment.
2. Hydrochloric acid, 0.1 N, diluted in LAL Reagent Water, for pH adjustment.
3. Water bath or heating block set at 37°C.
4. Spectrometer or filter photometer with a 545 nm filter.
5. Vortexer.
6. Timer.

Application Details

Comment:

Note: The dilution of standards and incubation temperature would be critical factors that can influence the absorption value, so it?s important to make sure that the endotoxin standard is fully dissolved, and the incubation temperature should strictly be kept at 37 °C.

Reagent Preparation:

Limulus Amebocyte Lysate (LAL)
Reconstitute lyophilized lysate by adding 1.7 mL LAL Reagent Water. Swirl each reconstitution gently for 30 seconds, avoid foaming. Reconstituted lysate remains stable if stored at -20°C for one week. For long-term usage, freeze the lysate at -80°C immediately after reconstitution. Avoid repeated freeze and thaw cycles.
Chromogenic Substrate
Reconstitute the substrate by adding 1.7 mL of LAL Reagent Water to a final concentration of ~2 mM. Once reconstituted, the substrate solution is stable for one month when stored at 2°C - 8°C. PROTECT SUBSTRATE FROM LONG-TERM EXPOSURE TO LIGHT
Stop Solution
Reconstitute the Color-stabilizer #1 (Stop Solution) with 10 mL of buffer S. The reconstituted Stop Solution is stable for one week when stored at 2°C - 8°C.
Color-stabilizer #2 and #3
Reconstitute Color-stabilizer #2 and #3 by adding 10 mL of LAL Reagent Water for both, respectively. Each reconstitution is stable for one week when stored at 2°C - 8°C. Endotoxin Standard Solution The amount of lyophilized endotoxin standard supplied in this kit is approximately 5 - 20EU. Please refer to the label on the vial of the endotoxin standard in the kit. Dissolve lyophilized endotoxin standard by adding 2 mL of LAL Reagent Water. Mix thoroughly for 15 minutes with a vortexer to obtain an endotoxin stock solution. The reconstituted endotoxin stock solution is stable for one week when stored at 2-8°C. DO NOT FREEZE THE ENDOTOXIN STOCK SOLUTION.
Prepare 1EU/mL endotoxin solution before making standard serial dilutions. For example, if the endotoxin stock solution is 5 EU/mL, dilute 0.2 mL of 5 EU/mL endotoxin stock solution with 0.8 mL of LAL Reagent Water to make the 1 EU/mL solution.
In each assay, at least four endotoxin standard solutions covering desired concentration range should be prepared to generate a standard curve. If the endotoxin concentration for the test sample is expected to be in the range of 0.01 - 0.1 EU/mL, the serial endotoxin standard solutions could be 0.1, 0.05, 0.025 and 0.01 EU/mL, respectively. If the endotoxin concentration in sample is expected in the range of 0.1 - 1 EU/mL, the serial endotoxin standard solutions could be 1, 0.5, 0.25 and 0.1 EU/mL, respectively.
An example of the preparation of serial endotoxin standard solutions is outlined in the figure below. Each solution should be mixed thoroughly for 30 seconds with a vortexer.

Sample Preparation:

All materials or diluents used for specimen collection and test reagent preparation must be endotoxin-free. Use aseptic technique at all times. Samples to be tested must be stored in such a way that all bacteriological activity is stopped or the endotoxin level may increase over time. For example, samples can be stored at 2-8 °Cwithin 24 hours before use, but need to be stored frozen if not used within 24 hours.
Dissolve or dilute test specimen using LAL Reagent Water. Since the LAL-endotoxin reaction is pH dependent, the pH value of the mixture should be between 6.0 and 8.0 to ensure standard and data linearity. Adjust the pH of the specimen with endotoxin-free 0.1N sodium hydroxide or 0.1N hydrochloric acid. Always measure the pH of an aliquot of the bulk sample, take care to avoid contamination by the pH electrode used. If the specimen contains interfering substances, dilute the specimen until that the interference is eliminated.

Assay Procedure:

1) Carefully dispense 100 µL of standard or test sample into endotoxin-free vials. All samples should be mixed thoroughly for 30 seconds with a vortexer. Avoid foaming/bubbles. Each test must include a blank as well as at least four endotoxin standards in duplicate. The blank sample vial contains 100 µL of LAL Reagent Water instead of test sample.
2) Add 100 µL of reconstituted LAL to each vial. Cap the vials and mix well by swirling gently.
3) If the endotoxin concentration in sample is expected in the range of 0.01 - 0.1 EU/mL, incubate the rack with all vials at 37°C±1°C for 45 minutes using a water bath or heating block. If the endotoxin concentration is expected in the range of 0.1 - 1 EU/mL, incubate at 37°C±1°C for 10 minutes.
4) After proper incubation, add 100 µL of reconstituted chromogenic substrate solution to each vial. Cap the vials and swirl gently to mix well. Do not shake or vortex to avoid foaming. Incubate at 37°C±1°C for 6 minutes.
5) Add 500 µL of reconstituted Color-stabilizer #1 (Stop Solution) to each vial and swirl gently to mix well. Do not shake or vortex to avoid foaming. Add 500 µL of reconstituted Color-stabilizer #2 to each vial and mix well. Finally add 500 µL of reconstituted Color-stabilizer #3 to each vial. Gently swirl each vial to mix well. Bubbles must be avoided.
6) Read the absorbance of each reaction vial at 545 nm using distilled water as blank to adjust the photometer to zero absorbance.

Calculation of Results:

Under the standard conditions, the absorbance at 545 nm shows a linear relationship with the concentration in the range of 0.01 to 1 EU/mL. Plot the absorbance for the four standards on the x-axis and the corresponding endotoxin concentration in EU/mL on the y-axis. Draw a best-fit straight line between these points and determine endotoxin concentrations of samples graphically.

Assay Precision: Linearity
The linearity of the standard curve within the concentration range used to predict endotoxin values need to be verified for each new lot of reagents. At least 4 endotoxin standards spanning the expected concentration range should be assayed along with a blank, in duplicate. The coefficient of correlation (r) for the individual mean absorbance of the standards vs. their corresponding endotoxin concentration should be ≥0.980.
Reproducibility
Replicate samples should be run in order to establish good technique and low coefficient of variation. The coefficient of variation (C.V.) equals 100 times the standard deviation of a group of values divided by the mean and is expressed as a percent. The C.V. absorbance should be less than 10%.

Restrictions: For Research Use only

Handling

Storage: RT/4 °C

Storage Comment: The kit can be stored dry at room temperature for up to 1 month. For long-term use, the kit can be kept at 2- 8 °C for up to one year. Do not freeze the kit or any of its components.

Expiry Date: 12 months

References

Findeisen, Voges, Braun, Sonnenberg, Schwarz, Körner, Reinecke, Sohrabi: "LXRα Regulates oxLDL-Induced Trained Immunity in Macrophages." in: International journal of molecular sciences, Vol. 23, Issue 11, (2022) (PubMed).

Schnack, Sohrabi, Lagache, Kahles, Bruemmer, Waltenberger, Findeisen: "Mechanisms of Trained Innate Immunity in oxLDL Primed Human Coronary Smooth Muscle Cells." in: Frontiers in immunology, Vol. 10, pp. 13, (2019) (PubMed).

Sohrabi, Lagache, Schnack, Godfrey, Kahles, Bruemmer, Waltenberger, Findeisen: "mTOR-Dependent Oxidative Stress Regulates oxLDL-Induced Trained Innate Immunity in Human Monocytes." in: Frontiers in immunology, Vol. 9, pp. 3155, (2018) (PubMed).

Shin, Takeda, Shiraishi, Jeong, Domenico, Jia, Han, Spallek, Singh, Lucas, Gelfand: "Microbial heat shock protein 65 attenuates airway hyperresponsiveness and inflammation by modulating the function of dendritic cells." in: Journal of immunology (Baltimore, Md. : 1950), Vol. 189, Issue 7, pp. 3404-10, (2012) (PubMed).

Murakami, Tanaka, Usui, Kawabata, Shiomi, Iguchi-Hashimoto, Shimizu, Yukawa, Yoshifuji, Nojima, Ohmura, Fujii, Umehara, Mimori: "Follistatin-related protein/follistatin-like 1 evokes an innate immune response via CD14 and toll-like receptor 4." in: FEBS letters, Vol. 586, Issue 4, pp. 319-24, (2012) (PubMed).

Caramelo-Nunes, Gabriel, Almeida, Marcos, Tomaz: "Purification of plasmid DNA from clarified and non-clarified Escherichia coli lysates by berenil pseudo-affinity chromatography." in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, Vol. 904, pp. 81-7, (2012) (PubMed).

Ahmad Khanbeigi, Kumar, Sadouki, Lorenz, Forbes, Dailey, Collins: "The delivered dose: Applying particokinetics to in vitro investigations of nanoparticle internalization by macrophages." in: Journal of controlled release : official journal of the Controlled Release Society, Vol. 162, Issue 2, pp. 259-66, (2012) (PubMed).

Joseph, Sambanthamoorthy, Dakshinamoorthy, Munirathinam, Ramaswamy: "Protective immune responses to biolistic DNA vaccination of Brugia malayi abundant larval transcript-2." in: Vaccine, Vol. 30, Issue 45, pp. 6477-82, (2012) (PubMed).

Sayeed, Nistico, St Croix, Di: "Multifunctional role of human SPLUNC1 in Pseudomonas aeruginosa infection." in: Infection and immunity, Vol. 81, Issue 1, pp. 285-91, (2012) (PubMed).

Sasidharan, Panchakarla, Sadanandan, Ashokan, Chandran, Girish, Menon, Nair, Rao, Koyakutty: "Hemocompatibility and macrophage response of pristine and functionalized graphene." in: Small (Weinheim an der Bergstrasse, Germany), Vol. 8, Issue 8, pp. 1251-63, (2012) (PubMed).

Medrano, Dolan, Condori, Radin, Cramer: "Quality assessment of recombinant proteins produced in plants." in: Methods in molecular biology (Clifton, N.J.), Vol. 824, pp. 535-64, (2011) (PubMed).

Target Details

Background: Genscript ToxinSensorTM Chromogenic LAL Endotoxin Assay Kit is designed to be a quantitative In Vitro end-point endotoxin test for human and animal parenteral drugs, biological products, and medical devices. This method utilizes a modified Limulus Amebocyte Lysate and a synthetic color producing substrate to detect endotoxin chromogenically in a broad range of 0.005 - 1 EU/mL. In addition, any sample with color (e.g. cell bacterial culture medium, serum or blood etc.) can not be assayed by this kit.