Endotoxins are small, stable, bacterially-derived hydrophobic molecules which can easily contaminate labware and whose presence can significantly impact both in vitro and in vivo experiments. Their presence is detected by the limulus amebocyte lysate (LAL) assay which can detect down to 0.01 Endotoxin Units (EU)/ml. Thorough cleanliness in labware, raw materials and in lab technique is required to substantially reduce endotoxin levels. Sigma’s ultrapure HyStem line of products is produced according to these guidelines, yielding a product that is suitable for demanding in vitro and in vivo experiments.
The original LAL/TAL method is the gel clot method. In this method equal amounts of a test sample, for instance an injectable chemotherapy solution and the gel clot LAL/TAL are mixed in a test tube. The combined solution is incubated at 37 °C for 60 minutes. After the incubation, the tube is inverted. If sufficient endotoxin is present in the test sample, the solution would have clotted during the hour incubation and a gel will remain in the bottom of the inverted tube (see image). If the sample does not contain detectable endotoxin, no clot will form and liquid will run down the side of the inverted tube. The gel clot method is still widely used throughout the world. It provides a yes/no answer to whether a sample contains a specified amount of endotoxin.
Additional LAL/TAL methods have been developed that are used to quantitate the amount of endotoxin in a test sample. The methods use software and instruments to monitor changes in the test sample/LAL/TAL mixture that are the result of endotoxin in the solution. Endotoxin standards at different concentrations are tested either at the same time as the test samples or in advance of them. The LAL/TAL response to the test samples is compared to the response to the endotoxin standards in order to determine the amount of endotoxin in the test sample.
Medicilon provides endotoxin testing for the pharmaceutical, biopharmaceutical and medical device industries.
In the kinetic turbidimetric LAL/TAL method, a 96-well plate reader or tube reader is used to monitor the change in solution clarity as a result of endotoxin present in a sample. The solution clarity or turbidity change is due to the formation of the gel in the well or tube. The faster the solution changes, the more endotoxin in the test sample or standard. The standard curve is plotted, typically on a log scale, as time (on the X-axis) and endotoxin concentration in Endotoxin Units (EU) or International Units (IU) per ml (on the Y-axis). The reaction time of the test sample is compared to the standard curve and an endotoxin concentration is calculated.
Two LAL/TAL methods use a chromogenic substrate to detect the presence of endotoxin in a solution. Rather than monitoring the change in solution turbidity, the kinetic chromogenic method monitors the change in color in a solution. More endotoxin, means the solutions in the well or tube turn yellow faster than solutions containing less endotoxin. The endpoint chromogenic method uses a fixed test time and the intensity of yellow color is used to distinguish between levels of endotoxin.
Endotoxin Detection Methods
1.Gel Clot LAL Assays: Qualitative Method
The PYROGENTTM Gel Clot LAL Assay is a qualitative LAL test for Gram-negative bacterial endotoxin. The gel clot assay is run in tubes that are placed in a water bath or dry heat block at 37ºC. After a one-hour incubation period, the tubes are flipped 180º. A firm clot that stays in the bottom of the tube indicates a positive reaction. If liquid flows down the side of the tube, the result is negative for endotoxin.
– Easy-to-read qualitative results
– Resistant to interference from glucans and pH
– Select from a wide range of kit sizes and sensitivities
2.Kinetic Turbidimetric LAL Assays: Quantitative Method
The PYROGENTTM-5000 Kinetic Turbidimetric LAL Assay is a quantitative method for the determination of Gram-negative bacterial endotoxin. The kinetic turbidimetric method is run on a 96-well plate at 37ºC in a microplate reader that measures absorbance at 340 nm. In the presence of endotoxin, the lysate
will begin to gel causing the solution to become cloudy or turbid. Higher concentrations of endotoxin cause the increase in solution turbidity to occur faster than lower concentrations.Typically, a standard curve is run with the unknown samples and the software calculates the amount of endotoxin in the unknown samples by comparing to the standard curve.
– Cost-effective method for water and large volume parenterals
– Sensitivity from 0.01 to 100 EU/ml
– Select from a wide range of kit sizes
3.Endpoint Chromogenic LAL Assays: Quantitative Method
The QCL-1000TM Endpoint Chromogenic LAL Assay was the first FDA-licensed endpoint quantitative LAL assay. The endpoint chromogenic method is run in tubes or on a 96-well plate incubated at 37ºC in a dry heat bath.
At T=0: Lysate is added to controls, standards and unknown samples
At T=10: Chromogenic substrate is added
At T=16: A stop reagent is added to stop the enzymatic reactions
The absorbance at 405-410 nm of the solutions in the tubes or 96-well plate is then read. With time, higher concentrations of endotoxin cause more yellow color to form (a higher absorbance value) than lower concentrations of endotoxin.Typically, a standard curve is run with the unknown samples and the amount of endotoxin in the unknown samples can be calculated by comparing to the standard curve.
– Less sensitive to product inhibition than assays requiring gel formation
– Sensitivity from 0.1 to 1 EU/ml
– Quantitative results in 16 minutes
4.Kinetic Chromogenic LAL Assays
Kinetic Chromogenic LAL Assays: Quantitative Method
The Kinetic-QCLTM Kinetic Chromogenic LAL Assay is a quantitative method for the detection of Gram-negative bacterial endotoxin. Like the endpoint chromogenic method, it is less sensitive to product inhibition than assays that require gel formation.The kinetic chromogenic method is run on a 96-well plate at 37ºC in a microplate reader that measures absorbance at 405-410 nm. In the presence of endotoxin, the lysate will begin to cleave the chromogenic substrate causing the solution to become yellow. Higher concentrations of endotoxin cause the increase in solution color to occur faster than lower concentrations. Typically, a standard curve is run with the unknown samples and the WinKQCLTM software calculates the amount of endotoxin in the unknown samples by comparing to the standard curve.
– Ideal for biological products such as vaccines and antibiotics
– Our most sensitive test with range from 0.005 to 50 EU/ml
5.Endpoint Fluorescent Assays: Quantitative Method
The PyroGeneTM Recombinant Factor C Endpoint Fluorescent Assay offers improvements for endotoxin detection testing.The PyroGeneTM Assay represents a reliable, sustainable alternative to LAL.
Lonza scientists have produced a recombinant form of FactorC, the first component in the horseshoe crab clotting cascade activated by endotoxin. Recombinant Factor C (rFC) is activated by endotoxin binding. The active enzyme then cleaves a synthetic substrate, resulting in the generation of a fluorogenic compound. The reaction is run in a 96-well plate and measured at time zero and after a one-hour incubation in a fluorescent microplate reader using excitation/emission wavelengths of 380/440 nm.
– Specific for endotoxin, eliminates positive glucan reactions
– Reliable lot-to-lot assay performance
– No animal utilization
– Comparable to other quantitative LAL methods
– Sensitivity range from 0.01 to 10 EU/ml
– FDA has approved 510(K) submissions using PyroGeneTM rFC Assay as a final release test
Medicilon’s Endotoxin Detection Testing Services offer you the best in routine and customized endotoxin testing. Our facilities are GMP compliant and provide expertise in gel clot, kinetic chromogenic, kinetic turbidimetric LAL, and rFC endpoint fluorescent assays.
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