PHERAstar FSX
Powerful and most sensitive HTS plate reader
Introduction
Small amounts of bacterial endotoxins, down to picograms or less than a billionth of a gram, are enough to trigger immune responses that lead to fever and severe adverse events if they are introduced into the body.1 Testing for bacterial endotoxins is a crucial step in the pharmaceutical and biotechnology industries to make sure that pharmaceuticals, biologics and medical devices do not unwittingly introduce these toxic substances into the body. The bacterial endotoxin test (BET test) is therefore a critical part of quality control testing to ensure that products introduced into the body do not instigate such adverse reactions.2-4
The BET test is one of several crucial methods developed to test for pyrogens, a diverse group of substances that lead to these unwanted, potentially life-threatening effects. Pyrogens can be either microbial in origin, such as bacterial endotoxins derived from Gram-negative bacteria, or arise from non-microbial sources. Other tests for endotoxins include the rabbit pyrogen test, the recombinant factor C test, and the monocyte activation test. Each of these tests is designed to ensure the safety of a wide range of products introduced into humans or other animals. Traditionally, the Limulus Amebocyte Lysate (LAL) assay is used for BET tests and plays a crucial role in many bioanalysis processes.2
In this blog, we look at the BET test or LAL test and examine how microplate readers can help support this test for endotoxins.
The BET test versus other pyrogen tests
The origins of the BET test began with fundamental research performed by Frederik Bang at the Johns Hopkins University School of Medicine back in the 1950s (Fig. 1).
Bang was the first to describe the toxic effect of a marine bacterium on Limulus polyphemus, a species of horseshoe crab, that involved the formation of blood clots.5 By 1956, he had described how the horseshoe crab could be used to study disease mechanisms.6 Bang went on to collaborate with Jack Levin to isolate the active clotting agent (amebocyte lysate) that would become the foundation of the first BET test namely the LAL assay.7-8 Their research was crucial in work towards determining the effectiveness of the BET test, and future efforts establishing criteria and processes to ensure compliance with guidelines for pharmaceutical and other products. Over time, different tests for pyrogens have been developed (Fig. 1) each with unique capabilities and features.
What is the BET test?
For many years, LAL has been the method traditionally used for BET testing. The BET test or LAL test refers to several methods that detect endotoxins from Gram-negative bacteria based on the clotting reaction of hemolymph isolated from different species of horseshoe crab. Hemolymph is akin to vertebrate blood. Amebocytes in hemolymph function as the horseshoe crab’s immune system (amebocytes are cells found in invertebrates that play a role in the defense against pathogens). If they encounter foreign substances like endotoxins, amebocytes generate clots that immobilize and kill invading pathogens. The ability of the LAL test to accurately detect endotoxins ensures the safety of various pharmaceutical products and medical devices.
The target for the BET test
As mentioned earlier, bacterial endotoxins are toxic molecules with serious repercussions for health and safety. They are widely distributed in the environment and amongst the most potent pyrogen s encountered.3,4 Specifically bacterial endotoxins are lipopolysaccharides derived from the cell wall of Gram-negative bacteria (Fig. 2).
Monitoring endotoxin contamination in medical devices and pharmaceuticals is crucial to ensure patient safety. This involves testing for bacterial endotoxins, which can induce fever and other severe reactions in humans.
Lipopolysaccharides are large molecules that are made up of lipid A (the toxic component responsible for most of the toxic effects of bacterial endotoxins), a core polysaccharide, and an O-antigen (a variable polysaccharide chain that helps the bacterium evade the host’s immune system). The purpose of the BET test is to provide a sensitive and accurate way to verify and, in most cases, quantify the presence of endotoxins.
Different sources of LAL for the BET test
Two species of horseshoe crab have been used as sources of LAL: Limulus polyphemus from the North Atlantic and Tachypleus spp. from Asia. Blood is collected in both cases from live animals and used as a source of an amebocyte-rich fraction. The amebocytes are lysed to release the coagulation proteins that are vital for the LAL test. Known endotoxin concentrations are used to standardize the LAL test by establishing a standard curve, which allows for accurate quantification of endotoxin levels based on the degree of light absorbance in the sample solution.
A clotting cascade
The immune response in Limulus occurs by a series of enzymatic reactions that are part of a complex clotting cascade (Fig. 3).
Factor C, the first enzyme in the series, binds to the hydrophobic lipid A component of the lipopolysaccharide molecule. This first step triggers a series of enzymatic reactions that lead to the formation of a blood clot. The BET test comprises this endotoxin-sensitive “tree” of clotting responses.
Various materials and conditions can cause inhibition in the LAL assay, affecting its ability to accurately detect endotoxins. Therefore, careful validation of the testing methods is necessary to ensure reliable outcomes in detecting endotoxins in pharmaceutical products and medical devices.
As shown in Fig. 3, the LAL assay can be triggered by two pathways: the factor C pathway and the factor G pathway. Both lead to the clotting enzyme response. The G pathway can be activated by β-glucans which can lead to false positive results in a LAL assay if β-glucans are present. Alternative tests are available that only have the factor C pathway, and which cannot be activated by β-glucans which gives them an advantage as an endotoxin test.
There are three main types of BET tests based on LAL methods: gel-clot; turbidimetric; and chromogenic assays. Here we will look at each one in a little more detail. Each BET or LAL test is simple and easy to perform, offers high sensitivity, and is cost effective. All these LAL tests are specific for bacterial endotoxins and do not detect non-endotoxin pyrogens (see Table 1).
Table 1. Features of LAL test versus other pyrogen/endotoxin tests.
| BET test or LAL test | Rabbit pyrogen test | rFC test | Monocyte Activation Test | |
| Type of pyrogen tested | Endotoxins (lipopolysaccharides) only | All pyrogens (endogenous and exogenous) | Endotoxins (lipopolysaccharides) only | Endotoxins and non-endotoxin pyrogens |
| Biological material or animal used | Amebocyte lysate from horseshoe crabs | Live rabbits | rFC | Human blood-derived monocytes, monocytic cell lines |
| Ethical concerns for animal use for test | Yes | Yes | No | No |
| Animal source of test components | Yes | Not applicable (animal is the test) | No | Yes (human blood-derived components) |
| Microplate assay | Yes | No | Yes | Yes |
| Detection mode | Absorbance, turbidimetric | Not applicable | Fluorescence (absorbance also an option for rCR) | Absorbance, fluorescence, luminescence |
| Sensitivity | High for endotoxins | Moderate for all pyrogens | High for endotoxins | High for all pyrogens |
| Specificity | Specific to endotoxins | Non-specific | Specific to endotoxins | Broad specificity |
| Regulatory status | Widely accepted (European, US, and Japanese Pharmacopeia | First test to be adopted and accepted for use by European and US Pharmacopeia | Accepted by European and US Pharmacopeia | Recognized by European Pharmacopeia for advanced applications |
Gel-clot BET test or LAL test
The gel-clot BET test involves visual inspection of gel formation. It is a qualitative test that provides a yes or no answer as to whether bacterial endotoxins are present in a sample. Bacterial endotoxin is a type of microbial pyrogen derived specifically from Gram-negative bacteria, and its detection is crucial in the pharmaceutical and medical device industries to ensure patient safety. The gel-clot BET test does not require a microplate reader or software for execution and can be readily performed in endotoxin-free test tubes or vials. The LAL reagent (freeze dried lysate from horseshoe crab blood) is incubated with a test sample at 37oC typically for 60 minutes. A positive result is indicated by a firm gel clot at the bottom of the tube or vial.
Turbidimetric BET test or LAL test
The turbidimetric BET test or LAL assay reveals not only the presence of endotoxin but also quantifies the amount present. The assay depends on measuring the amount of light scattering at 340 nm due to the presence of the LAL clot. The microplate reader measures the reduction in transmitted light caused by the scattering by means of absorbance. The results are calculated from a standard curve.
The turbidimetric BET test measures the increase in turbidity (cloudiness) that occurs when the LAL reagent reacts with endotoxin. Two types of turbidimetric BET tests are possible: namely kinetic and endpoint assays. In a kinetic turbidimetric method, the rate of turbidity increase is measured, which relies on spectrophotometry to measure the light absorbance in a sample solution. This method is effective in determining endotoxin levels but can be compromised by the presence of color, insoluble particles, or high viscosity in samples. In an endpoint assay, the turbidity is measured at a fixed time point. The turbidity can be measured in a microplate reader by measuring the absorbance at 340 nm. Samples are incubated at 37oC. Microplates should be endotoxin free and clear, flat bottom plates are needed for absorbance measurements. Non-binding or low-protein binding surfaces are recommended.
The use of a BMG LABTECH microplate reader for turbidimetric readouts of light scattering (absorbance mode) is described in the application note Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay. The PYROSTAR ES-F/Plate LAL test is based on the classic LAL assay principle and is sensitive, robust, and suitable for small sample volumes. It is not activated by β-glucans which can lead to false positive results.
Chromogenic BET test or LAL assays
In the chromogenic BET test or LAL assay a quantitative readout of the concentration of endotoxin in the sample is produced due to color development. The LAL reagent is mixed with a chromogenic agent (e.g. a peptide connected to p-nitroaniline) to produce a synthetic chromogenic substrate. The substrate is added to the test sample to create a test solution which is incubated at 37oC. The incubations can be performed on endotoxin-free microplates. In the presence of endotoxins, the peptide bonds connecting the peptide to p-nitroaniline are broken releasing the yellow color into solution. The amount of endotoxin is measured by monitoring the absorbance increase at 405-410 nm.
It is crucial to evaluate the effectiveness of the chromogenic method to ensure its reliability and accuracy in detecting endotoxins.
Alternatives to reduce the environmental impact of the BET test or LAL test
The use of animals as a source of proteins for the LAL test has affected the overall population of horseshoe crabs in the wild particularly in Asia. In the United States, several conservation efforts have demonstrated ways to sustain horseshoe crab populations but the development of alternative tests like the recombinant factor C (rFC) test that avoid harvesting horseshoe crab blood from live animals avoid this environmental impact.
The toxicity of bacterial endotoxins, particularly the Lipid A component of lipopolysaccharides found in Gram-negative bacteria, poses significant risks in medical devices and pharmaceuticals. Reducing endotoxin contamination is crucial to prevent severe patient harm, highlighting the importance of rigorous testing and monitoring protocols.
BET tests on a microplate reader
BET tests benefit from the consistent, cost-effective and higher throughput analysis that microplate readers can deliver. As mentioned in the previous section, absorbance or turbidimetric detection are the methods of choice to determine endotoxin levels using the BET test (LAL assay). Detecting these endotoxins is crucial because these toxins can lead to harmful febrile reactions in humans, making it essential to ensure their removal to maintain product safety.
Here we demonstrate applications for BET tests on microplates and highlight distinctive features of BMG LABTECH solutions.
In the application note Analysis of endotoxin concentrations using the absorbance detection mode the use of absorbance-based readouts for turbidimetric and colorimetric assays are described. The PYROSTAR ES-F/Plate LAL test and Limulus Color KY test from Wako were used for the turbidimetric and colorimetric assays, respectively. LAL clotting and endotoxin concentrations can be readily quantified using either test. In the PYROSTAR ES-F/Plate LAL test light scattering due to endotoxins is measured as an absorbance readout. In the Limulus Color KY test the increase in absorbance due to the increase in p-nitroaniline is quantified by the microplate reader.
In the application note Endotoxin detection on a microplate reader using a colorimetric, kinetic endotoxin detection kit with integrated data analysis the use of a LAL assay is described for the quantification of bacterial endotoxins. The Lonza Kinetic QCL™ was used together with either a filter-based or monochromator-equipped microplate reader from BMG LABTECH to perform the BET test. The assay relies on measuring the release of p-nitroaniline from the colorless peptide Ac-Ile-Glu-Ala-Arg-pNA. The liberation of p-nitroaniline results in a yellow color which is measured at 405 nm. The concentration of bacterial endotoxin was readily calculated by reference to a standard curve (Fig. 4).
The use of the MARS data analysis package allows for easy handling and data processing of the results generated from endotoxin measurements (Fig. 5).
Clotted LAL and endotoxins can also be measured using nephelometry by measuring forward scattered light. The use of the NEPHELOstar Plus for light scattering measurements is described in the application note Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay. The PYROSTAR™ ES-F/Plate LAL is also suitable for spectrometer-based measurements of endotoxins as we saw earlier.
Alternatives to BET tests
BET or LAL tests have traditionally been used for many products arising from biotechnology, drug discovery, microbiology, immunology and many other areas of quality control and bioanalysis. The availability of new tests such as the monocyte activation test or recombinant factor C test will provide further impetus and options to ensure safety across different industries in the life sciences.
Ensuring products are non-pyrogenic will remain crucial in medical device and pharmaceutical manufacturing. Products labeled as non-pyrogenic undergo specific testing to ensure they do not release fever-inducing substances, thereby maintaining patient safety during medical procedures.
BMG LABTECH solutions
What is the preferred BMG LABTECH microplate reader for specific needs and applications related to the determination of bacterial endotoxins? BMG LABTECH offers a range of absorbance microplate readers for sensitive measurements of BET tests. These include the SPECTROstar Nano, SPECTROstar Omega, CLARIOstar® Plus, VANTAstar® and PHERAstar® FSX. The SPECTROstar Nano is a single-mode dedicated absorbance plate reader. All other readers have multi-mode detection capabilities, including for instance fluorescence and luminescence detection to cover other pyrogen tests such as rFC and monocyte activation tests.
BMG LABTECH readers are equipped with a UV/vis spectrometer for absorbance detection. This ensures wavelength flexibility in absorbance assays and fast spectral scanning capabilities. The spectrometer can measure any wavelength from 220-1000 nm or provide a full spectrum in this spectral range in less than 1 second per sample. The CLARIOstar Plus and VANTAstar additionally offer outstanding wavelength flexibility in fluorescence and luminescence, which is an asset for other assays. Increased light transmission and sensitivity is possible courtesy of Linear Variable Filter MonochromatorsTM and different filter options.
The NEPHELOstar® Plus is a dedicated microplate nephelometer that detects insoluble particles in liquid samples by measuring forward scattered light. Its high-intensity 635-nm laser light source is suitable for sensitive, quantitative, real-time measurements of turbidity at high throughput. The NEPHELOstar Plus is a great option for turbidimetric LAL kits where users may require less background noise and better quality data through the direct measurement of scattered light in contrast to the indirect measurement on absorbance-based readers.
Additional microplate reader features like incubation and shaking provide further benefits for BET tests. The enzymatic cascades that are the central element of the assay have an optimum temperature of 37°C. Only a temperature incubation option makes it possible to monitor the signal development of the assay over time. All BMG LABTECH readers offer accurate temperature regulation up to 45°C (optionally up to 65°C). The available shaking options support users in the proper mixing of assay reagent before the kinetic monitoring starts.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus, and PHERAstar FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection. The VANTAstar can be equipped with a modular injection unit. These readers readily support the different and emerging methods for endotoxin detection, including the kinetic turbidimetric method. The MARS data analysis package is provided with all BMG LABTECH microplate readers.
Collectively, BMG LABTECH multi-mode readers combine high-quality measurements with miniaturised assays, short measurement times, and offer considerable savings on materials and other resources.
References
- Iwanaga S. Biochemical principle of Limulus test for detecting bacterial endotoxins. Proc Jpn Acad. Ser. B Phys. Biol. Sci. 2007 83(4): 110-119. doi: 10.2183/pjab.83.110.
- Schneier M, Razdan S, Miller AM, Briceno ME, Barua S. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Biotechnol. Bioeng. 2020 117(8): 2588-2609. doi: 10.1002/bit.27362.
- Magalhães PO, Lopes AM, Mazzola PG, Rangel-Yagui C, Penna TC, Pessoa A Jr. Methods of endotoxin removal from biological preparations: a review. J. Pharm. Pharm. Sci. (2007) 10(3): 388-404.
- Bang FB. The toxic effect of a marine bacterium on Limulus and the formation of blood clots. Biol. Bull. (1953) 105:447-448.
- Bang FB. A bacterial disease of Limulus polyphemus. Bull. Johns Hopkins Hosp. (1956) 98:325.
- Levin J and Bang FB. A description of cellular coagulation in Limulus. Bull. Johns Hopkins Hosp. (1964) 115:337.
- Levin J and Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. (1964) 115:265.6.
- Levin J, Bang FB. Clottable protein in Limulus: Its localization and kinetics of its coagulation by endotoxin. Thromb. Diathes. Haemorrh. (Stuttg) (1968) 19:186.
- Ding JL, Ho B. Endotoxin detection--from limulus amebocyte lysate to recombinant factor C. Subcell. Biochem. (2010) 53:187-208. doi: 10.1007/978-90-481-9078-2_9.
