Microplate Reader Innovations To Accelerate Pyrogen Detection

Pyrogen and endotoxin tests: Past, present and future Pyrogen and endotoxin tests: Past, present and future 2 FOREWORD Contamination of pharmaceuticals, biotherapeutics or medical devices with pyrogens can cause elevated temperature and fever with potentially serious risks to health. The rapid and reliable detection of pyrogens using highly sensitive tests is therefore vital for health and safety. Pyrogens include many diverse molecules and other substances that make the requirement of excluding them from manufacturing processes and final products challenging. Non-endotoxin pyrogens range from components of Gram-positive bacteria, yeast, molds and viruses to environmental particles like rubber, plastic, and dust. Endotoxin pyrogens consist of molecular triggers within the lipopolysaccharides of frequently encountered Gram-negative bacteria. Irrespective of origin, pyrogens need to be carefully detected and prevented from interacting with the body’s immune system. Innovation is expanding the testing options available to ensure the pyrogenfree status of products. This progress reflects the move away from animalbased tests, advances in science and technology, and important changes in the regulatory environment. This eBook explores some of the different ways to detect pyrogens and how microplate readers can be used to support these crucial tests for quality control. Learn more inside. Dr. Barry Whyte Application Scientist and Science Writer Pyrogen and endotoxin tests: Past, present and future 3 CONTENTS INTRODUCTION Pyrogens and endotoxins: the importance of excluding life-threatening substances from health interventions 4 MICROPLATE READERS AND PYROGEN TESTING The roles of microplate readers in pyrogen testing 13 A DIFFERENT ANGLE Luca Di Bello from bioMérieux shares insights on sustainable endotoxin testing and microplate reader requirements 18 CODA Industry-wide applications for pyrogen tests 21 REFERENCES 22 APPLICATION NOTES Endotoxin detection on a microplate reader using a colorimetric, kinetic endotoxin detection kit with integrated data analysis 25 Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay 27 Colorimetric and turbidimetric analysis of endotoxins using the absorbance detection mode 29 Faster PyroGene™ Detection of Endotoxin using Enhanced Dynamic Range on the CLARIOstar Plus 31 Stream-lined detection of endotoxin using the ENDOZYME® II recombinant Factor C assay and Enhanced Dynamic Range on the VANTAstar® 33 Fast and highly sensitive pyrogen detection with the LumiMAT™ assay performed on BMG LABTECH microplate readers 35 Multiplex analysis of inflammatory cytokines from primary human macrophages using a FLUOstar® Omega 37 Highly sensitive ELISAs in 90-minutes: SimpleStep ELISA® kits and the SPECTROstar® Nano 39 CONCLUSION 41 Pyrogen and endotoxin tests: Past, present and future 4 Pyrogens and endotoxins: the importance of excluding life-threatening substances from health interventions INTRODUCTION What are pyrogens? Pyrogens are a broad range of substances that can produce an immune response that subsequently triggers an increase in temperature in a human or other animal. 1 They can be grouped into two main categories: exogenous and endogenous. Exogenous pyrogens arise from outside the body and induce fever reactions after administration. These types of pyrogens may accompany pharmaceuticals and biotherapeutics when they are introduced into the body by injection or infusion or when medical devices are used as health interventions. In contrast, endogenous pyrogens such as interleukins or tumor necrosis factor alpha (TNFα) are produced by the body itself as a reaction to contact with exogenous pyrogens. Pyrogen tests are of paramount importance in assuring health and safety when materials are introduced into the body since the presence of pyrogens can lead to an immune response with fever and serious adverse events. In addition to contamination, even products themselves, for example interleukins used as drugs for cancer treatments, can act directly as pyrogens. A further useful distinction for the diverse substances that make up pyrogens is classification into endotoxin and non-endotoxin pyrogens (Table 1).2-5 Table 1. Diversity of pyrogens. SOURCE EXAMPLES Non-endotoxin pyrogens Gram-positive bacteria Lipoteichoic acid Yeast Zymosan Viruses Double-stranded RNA Environmental particles Rubber, plastic Endotoxin pyrogens Gram-negative bacteria Lipopolysaccharide Non-endotoxin pyrogens range from components originating from Gram-positive bacteria, yeasts, molds and viruses to Pyrogen and endotoxin tests: Past, present and future 5 Figure 1: Lipid A endotoxin from Escherichia coli K12. This figure is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported licenses and was adapted from: http://www.jlr.org/content/47/5/1097.full. Figure 3: The toll-like receptor family. Figure 2: The bacterial cell wall. Pyrogens and the immune system The primary interface for pyrogens and the triggering of fever in the body is through activation of the innate immune system. The activation of the innate immune system arises through interactions with toll-like receptors (TLRs) on monocytes (figure 3). Monocytes are a type of white blood cell (leukocyte) that can differentiate within tissues into macrophages and monocyte-derived dendritic cells. These cells either eliminate the pathogen or alert other immune cells to help destroy the invading infectious organism and prevent infection. Toll-like receptors are pattern recognition receptors that play a crucial role in detecting pathogens and activate the immune response through the production of endogenous pyrogens such as cytokines. Toll-like receptors recognize pathogen-associated molecular patterns (PAMPs; e.g. glycans, flagellin, dsRNA) from microbes including viruses and fungi and damage-associated molecular patterns arising from injured cells. They also recognize dangerassociated molecular patterns (DAMPs) which are endogenous molecules that are released from dying cells. When toll-like receptors are stimulated by PAMPs or DAMPs different signaling cascades are initiated that give rise to specific immunological responses. In most cases, the different toll-like receptors on the cell surface use myeloid differentiation primary-response protein (MyD88) as one of the first proteins in the cascade of signaling reactions that are possible within the cell. Ultimately, the different signaling pathways, which include those originating from within the cell (for example from the endosome), converge on the regulation of different environmental particles like rubber, plastic, dust and packaging materials.2-4 Endotoxins include components of lipopolysaccharide (LPS) that originate from Gram-negative bacteria, microorganisms that are widely encountered in the environment. The toxic component of bacterial endotoxin is lipid A (figure 1), one of the components of the lipopolysaccharide present in the bacterial cell wall (figure 2). Lipid A is a phosphorylated N-acetylglucosamine disaccharide containing a hydrophobic region (comprised of the aliphatic chains of fatty acids). The hydrophobic domain anchors the endotoxin into the bacterial membrane. The remaining parts of the endotoxin, which include the core polysaccharide and O-antigen, are projected from the cell surface. The O-antigen is attached to the core polysaccharide and is the outermost part of the molecule. While the O-antigen is not toxic, it is the main immunogenic portion of the endotoxin and serves as a recognition target for antibodies. It is the most diverse component of lipopolysaccharide, and its composition and length vary considerably among different bacterial species and even within strains of the same species of bacteria. Pyrogen and endotoxin tests: Past, present and future 6 Invasion/Application Adjustment of body temperature set point Fever Cells of the innate immune system monocytes Exogenous pyrogens: Microbial contaminants Monocyte activators Endogenous pyrogens: Interleukin 1β, interleukin 6, tumor necrosis factor α Figure 4: The link between pyrogens, the immune system and fever. transcription factors in the nucleus that lead to the production of cytokines like TNFα and the different interleukins. Monocytes activated in this way secrete cytokines (endogenous pyrogens) that travel through the bloodstream to the brain where they can stimulate the production of prostaglandins (figure 4). The binding of prostaglandin to cell receptors in the hypothalamus leads to an increase in the body’s thermoregulatory set point. This interaction sets in motion the body’s heat generating and heat conservation mechanisms that are a feature of the fever response. In some cases, these changes are life threatening. Toll-like receptors and different pyrogens Toll-like receptors are a diverse group of proteins that interact with a wide range of ligands. This diversity means that the different types of pyrogens can trigger increases in cytokines through many distinct signaling pathways within the cell. Typically, bacterial cell wall components are recognized by tolllike receptors on the cell membrane. Nucleic acids are recognized by intracellular toll-like receptors that are present on the surface of endosomes within the cell. What does this mean for the different groups of substances that make up pyrogens? First and foremost, it means that there are many routes to triggering the human fever reaction in the hypothalamus. Exogenous endotoxins like lipid A can bind to toll-like receptor 4 (TLR4) on the cell membrane to trigger a signaling cascade that promotes endogenous cytokine release from within the cell (IL-1, IL-6 or TNFα). Nonendotoxin pyrogens like lipoteichoic acid or double-stranded RNA can bind to specific toll-like receptors either on the surface of the cell or to toll-like receptors present within the cell also triggering immune cell activation. All these events can trigger cytokine release, immune cell activation, and stimulation of the hypothalamus leading to fever. The importance of pyrogen testing The detection of endogenous and exogenous pyrogens is of great importance to patient safety and is closely regulated by standards and organizations including the Food and Drug Administration (FDA), United States Pharmacopeia (USP) and the European Pharmacopeia (EP). Contamination of pharmaceuticals, biotherapeutics or medical devices with pyrogens can cause elevated temperature and fever in patients with potentially serious risks to health and quality control is needed across many manufacturing Pyrogen and endotoxin tests: Past, present and future 7 Rabbit Pyrogen Test 1912 LAL Test 1956 Monocyte Activation Test 1995 Recombinant Factor C 2001 rFC rFC‘ A Critical Study of Experimental Fever. By Edward C. Hort, F.R.C.P. Edin, and W.J. Penfold, M.B., C.M. (Communicated by Dr. C.J. Martin, F.R.S. Received February 17,-Read March 14, 1912) (From the Lister of Preventive Medicine.) METHODS TO DETECT PYROGENS COMPLETE PYROGEN TESTS Test type RPT Rabbit Pyrogen Test MAT Monocyte Activation Test LAL Limulus Amebocyte Lysate Test rFc Recombinant Factor C Test USP <151> Japanese Pharmacopeia Ph. Eur. 2.6.30 Ph. Eur. 2.6.14 <USP 85> Japanese Pharmacopeia Ph. Eur. 2.6.32 USP <86> Raise of body temperature after injection of drug (in vivo) Inflammatory cytokines released from human monocytes (human blood-derived monocytes, monocytic cell lines) Clotting cascade in blood amebocyte lysate from horseshoe crabs (in vitro) Recombinant Factor C (first step in LAL cascade) (in vitro) Detects endotoxins Detects non-endotoxin pyrogens Animal free Regulatory compliance Measuring mechanism Temperature Absorbance, fluorescence, luminescence Absorbance, turbidity Fluorescence (absorbance also an option for recombiant Cascade Reagent rCR) Detection mode Sensitivity Moderate for all pyrogens High for all pyrogens High for endotoxins High for endotoxins Specificity Non-specific Broad specificity Specific to endotoxins Specific to endotoxins BACTERIAL ENDOTOXIN TESTS Figure 5: Timeline for development of pyrogen and endotoxin tests. Figure 6. Methods to detect pyrogens. processes. Over the years, a variety of tests have been developed to detect different factors associated with pyrogens. Pyrogen testing is essential for the manufacture of vaccines and biotherapeutics, the preparation of parenteral pharmaceuticals, cell and gene therapies, the production of recombinant proteins and nucleic acids, and the medical device industry. The rapid, reliable detection of pyrogens using highly sensitive tests is therefore vital to ensure health and safety. Over time, different tests for pyrogens have been developed (figure 5) each with unique capabilities and features. Four main methods are currently available for pyrogen tests and endotoxin detection (figure 6).2,3 They are distinguished by whether they use animals for testing and their target. The target can either be (a) exclusively endotoxins or (b) pyrogens more widely (defined as endotoxins and nonendotoxin pyrogens). The rabbit pyrogen test The first fever-causing agents were identified by Hort and Penfold at the Lister Institute of Preventive Medicine in 1912.6 Hort and Penfold were the first to design a pyrogen test based on the injection of substances into rabbits which is the foundation of today’s rabbit pyrogen test. The rabbit pyrogen test is a “broad” pyrogen testing method that can detect both endotoxin and non-endotoxin pyrogens. The test relies on the measurement of body temperature in rabbits after injection of the product into the ear vein. A rise in body temperature indicates the presence of pyrogens in the injected sample. While the rabbit pyrogen test has been widely used over the years, it is not a quantitative assay and has low sensitivity. This test is designed to limit the risk of a fever reaction to an acceptable level when the product is introduced into the patient. A significant drawback is the need for large numbers of Pyrogen and endotoxin tests: Past, present and future 8 animals to perform pyrogen tests and the inevitable suffering that accompanies this approach. The bacterial endotoxin test The bacterial endotoxin test (BET test) is a method to detect components of the outer membrane of Gram-negative bacteria (specifically lipopolysaccharides) that can cause fever and adverse events when introduced into the body. Unlike the rabbit pyrogen test, the BET test does not detect non-endotoxin pyrogens. 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.7 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 (figure 5). 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.8 By 1956, he had described how the horseshoe crab could be used to study disease mechanisms.9 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 (Limulus Amebocyte Lysate) assay.9-11 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. For many years, LAL has been the method traditionally used for BET testing. It 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. As mentioned earlier, bacterial endotoxins are toxic molecules with serious repercussions for health and safety. Specifically bacterial endotoxins are lipopolysaccharides derived from the cell wall of Gram-negative bacteria. 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. Two species of horseshoe crab have been used as sources of LAL: Limulus polyphemus from the North Atlantic and Tachypleus spp. from Asia. In both cases, blood is collected 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 transmittance through the sample solution. The immune response in Limulus occurs by a series of enzymatic reactions that are part of a complex clotting cascade (figure 7). B-1,3-Glucan Endotoxin Factor C Factor C‘ Factor B Factor B‘ Factor G‘ Factor G Coagulogen Coagulin (gel clot) Turbidimetric Chromogenic substrate Color development (yellow) Proclotting enzyme Clotting enzyme Figure 7: The bacterial endotoxin test or LAL test. 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. Pyrogen and endotoxin tests: Past, present and future 9 As shown in figure 7, 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 utilise the factor C pathway, and which cannot be activated by β-glucans which gives them an advantage as an endotoxin test. 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. 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 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 nonendotoxin pyrogens. Gel-clot 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. The gel-clot BET test does not require a device 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 37 °C typically for 60 minutes. A positive result is indicated by a firm gel clot at the bottom of the tube or vial. Turbidimetric 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 due to the presence of the LAL clot at a specific wavelength (depends on manufacturer) . A spectrophotometer or a microplate reader can be used to measure 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 transmittance through 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 by measuring the light transmittance at a specific wavelength. Samples are incubated at 37 °C. Endotoxin free tubes, vials or microplates (clear, flatbottom) are needed for measurements on a spectrophotometer or absorbance microplate reader. Non-binding or low-protein binding surfaces are recommended. Since turbidimetric assays depend on determining the amount of light scattering, measurements are also possible on a nephelometer such as the NEPHELOstar Plus with kits like the PYROSTAR™ ES-F series. This endotoxin detection kit is available from FUJIFILM Wako and is compliant with United States Pharmacopeia recommendations for endotoxin assays. The reagents in this kit cannot be activated by β-glucans which helps to avoid false positive results. You can learn more about the use of nephelometry and the PYROSTAR™ ES-F series kit in the application note Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay. (page 27) Chromogenic test 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 with no clot generation. 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 37 °C. The incubations can be performed on endotoxinfree tubes or 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 Pyrogen and endotoxin tests: Past, present and future 10 endotoxin is measured on a spectrophotometer or microplate reader 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 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.12 The recombinant factor C test The recombinant factor C (rFC) test is an in vitro method used to detect bacterial endotoxins. It is usually based on a fluorescent readout but absorbance detection is also possible for recombinant Cascade Reagent (rCR), an optimized kinetic chromogenic reagent that simulates the natural LAL reaction.1,2 rFC is a genetically engineered protein that is activated by endotoxin. Once activated, the rFC enzyme typically releases a fluorescently labeled product from an engineered substrate that is quantifiable and proportional to the amount of endotoxin present. It serves as an alternative to the LAL assay and plays a crucial role in many quality control and bioanalysis processes.12-14 As such, it provides a more sustainable alternative to traditional animal tests or animalderived test components. The adoption of rFC in several industries highlights its benefits in ensuring patient safety and its alignment with animal conservation efforts. In an rFC test, a genetically engineered protein, which is modelled on the natural protein from the horseshoe crab, is activated by endotoxin to produce a fluorescent product from a substrate that is easily quantified (figure 8). When lipopolysaccharide binds to rFC, it activates the protease activity of the enzyme. The assay includes a synthetic fluorogenic substrate which is cleaved by activated rFC to release the fluorescent tag. The amount of fluorescence is directly proportional to the amount of endotoxin present. The main counterpart of the rFC test in the pyrogen testing family is the LAL assay. The rFC test essentially utilizes a recombinant form of the Limulus clotting factor, which is crucial for detecting bacterial infections. Both the LAL and rFC tests rely on factor C as part of the detection process. Unlike the LAL test, the rFC test only detects the factor C pathway and does not impact the factor G pathway. It therefore cannot be activated by β-glucans which gives it an advantage as an endotoxin test. Unlike the rFC test which relies on fluorescence measurements, detection of bacterial endotoxins with the LAL test is typically either by absorbance or turbidimetric measurements. Methods like the rFC test that do not depend on animal sources are attractive alternatives to the LAL test. The rFC test was accepted in the US Pharmacopeia and was officially recognized in May 2025. It has been available in the European Pharmacopeia since 2020 and permitted for use since its inclusion. Figure 8: Recombinant factor C (rFC) assay. Advances in rFC testing Traditional endotoxin testing methods often require heavy manual preparation leading to inefficiencies, increased costs, and inaccurate results. rFC tests offer significant improvements in efficiencies, savings on resources, and improved sensitivity for measurements. Further advances in rFC tests will continue to improve performance and increase throughput. The ENDOZYME® II GOPLATE™ from bioMérieux is one example where the availability of a ready-to-go microplate precoated with standard and positive controls eliminates the need for dilution and reconstitution steps. The ability to streamline workflows by eliminating unnecessary steps Pyrogen and endotoxin tests: Past, present and future 11 offers savings in time and resources. At the same time, it makes automation easier. The adoption of rFC as a reagent for detecting bacterial endotoxins is poised for further growth as choice of suppliers increases and awareness of the favorable regulatory environment improves. Other options like Endosafe® Trillium™ recombinant Cascade Reagent (rCR) from Charles River Laboratories offer further choices for sustainable endotoxin testing. rCR is an optimized kinetic chromogenic reagent curated to simulate the natural LAL reaction. Trillium includes three critical biological proteins (recombinant Factor C, recombinant Factor B, and recombinant proclotting enzyme) and a specific concentration of key components. rFC is an endpoint method and only contains a single protein in the cascade. Trillium has been specifically developed to provide the highest quality results among recombinant technologies. Its proprietary matrix demonstrates improved accuracy and robustness compared to other recombinant endotoxin detection technologies and primary validation data in water support the claim of equivalency to LAL. The assay uses a chromogenic substrate that releases a yellowcolored product (p-nitroaniline) when cleaved by the final activated enzyme in the rFC cascade. Detection is by absorbance measurements. Further benefits will arise if ways are found to extend the range of applicability of recombinant proteins for endotoxin testing. The EndoLISA® test from bioMérieux for example includes a novel phage-derived receptor protein with high affinity and specificity for lipopolysaccharide. After sample binding, a wash step permits the elimination of interfering components like proteins, detergents or salts which makes the subsequent detection by recombinant factor C more robust and reliable. While this test is still not suitable for the detection of endotoxins in blood samples, its enhanced properties make it a more robust assay for testing many recombinant proteins, monoclonal antibodies or vaccines. The test is often used to ensure materials like viral vectors used in cell and gene therapies are endotoxin free. The monocyte activation test The monocyte activation test (MAT) is a cellbased, in vitro assay for pyrogens that is an alternative to traditional animal testing methods like the rabbit pyrogen test. 16, 17 It plays an important role in many quality control and bioanalysis processes for the manufacture and batch testing of drugs and medical devices due to its ability to test for endotoxin and nonendotoxin pyrogens. For example, the monocyte activation test is often used to ensure the production and safety of vaccines, antibiotics and plasma-derived drugs. 3 When monocytes are activated by pyrogens, they produce cytokines (e.g. interleukins or TNFα). These molecules can be used as indirect readouts for the presence of pyrogens and can be detected by different types of immunological assays including Enzyme-Linked Immunosorbent Assays (ELISAs) and other types of immunoassays that offer sensitive, quantitative measurements of cytokines. Cytokines can also be detected using gene reporter assays which offer a rapid read out, wide dynamic range and simplified protocol. Monocytes are a good option as a detection test for pyrogens due to their role in the immune response in humans. The test essentially leverages the role that monocytes play in innate immunity (see Introduction). When pyrogens activate monocytes in the innate immune system, the cells secrete pro-inflammatory cytokines. These readily generated cytokines can be tested as proxies for pyrogen contamination in different in vitro assays at high sensitivity under conditions suitable for a reliable, fast testing method. Typically, in a monocyte activation test a parenteral is incubated with monocytes (cell line or peripheral blood mononuclear cells). After incubation (generally overnight) the sample is tested for cytokine presence by ELISA. The test offers a low limit of detection that is crucial for product-specific validation. The main counterpart of the monocyte activation test in the pyrogen testing family is the rabbit pyrogen test. However, the rabbit pyrogen test sometimes lacks reproducibility and accuracy. It is also relatively expensive and raises ethical concerns due to the use of live animals. As an in vitro alternative, the monocyte activation test offers several benefits over traditional methods. It provides improved accuracy, good sensitivity, and does not require the use of lab animals, aligning with current regulatory recommendations and advancements in the field. Regulatory organizations and guidelines such as the European Pharmacopeia have advocated Pyrogen and endotoxin tests: Past, present and future 12 for the adoption of in vitro alternatives like the monocyte activation test in place of traditional animal-based tests. Hundreds of thousands of rabbits are used worldwide each year for pyrogen testing and alternatives are needed. The European Pharmacopeia General Chapter has for some time suggested replacing the rabbit pyrogen test with the monocyte activation test as a validated approach to ensure products are labeled ‘pyrogen free’. In June, 2024, the European Pharmacopeia Commission removed the rabbit pyrogen test from its monographs and mandated the adoption of non-animal methods like the monocyte activation test. The European Pharmacopeia Commission abolished the use of the traditional rabbit pyrogen test from 1 July 2025. 18 While progress is being made to reduce the number of animals used in testing, animal use remains considerable worldwide despite the availability of alternative tests. Further developments in regulatory requirements coupled with ongoing innovation in testing methods should help reduce unwanted animal tests. Different monocyte activation test kits are available commercially, which can be categorized into two main types based on their cell sources: those utilizing cell lines and those relying on donor-pooled peripheral blood mononuclear cells. Both cell sources effectively detect endotoxins and non-endotoxin pyrogens. For monocyte activation tests, absorbance, fluorescence intensity and luminescence measurements are all methods of choice to determine pyrogen levels accurately. In most cases, absorbance or fluorescence reading modes are typically used for detection. MAT gene reporter assays Although less common, luminescence readout is an option for some types of ELISA assays. Luminescence is also used to measure NF-κB activity using gene reporter assays which are a good, quick, sensitive one-step alternative to conventional monocyte activation tests. Gene reporter techniques rely on the use of a reporter gene typically placed downstream of a regulatory sequence. If the regulatory sequence is activated or repressed the reporter gene is expressed at different levels and the product can be measured quantitatively using different detection techniques. The LumiMAT™ assay from FUJIFILM Wako uses the monocytic cell line NOMO-1 in which the luciferase reporter gene has been introduced to express luciferase protein in response to NF-κB activation when these cells are triggered by pyrogens (figure 9). LumiMAT makes use of a convenient reporter assay in a stable reporter cell line. This offers a stable supply of cells, good reactivity and less lot-to-lot variability. Significantly, the LumiMAT assay allows rapid testing in around 5 hours (compared to more than 12 hours for a regular MAT assay combined with an ELISA readout). LumiMAT therefore offers a faster readout, higher sensitivity, wider dynamic range and a simplified protocol compared to other monocyte activation tests. MyD88 MyD88 MyD88 MyD88 MyD88 MyD88 TRIF MyD88 TRIF MyD88 NF-κB NF-κB Extracellular TLR1/2 TLR2/6 TLR4 TLR3 TLR9 TLR7 TLR8 TLR5 Intracellular Substrates Luminescence Nuclear Translocation Endosome Inflammatory cytokines (IL-1β, IL-6, TNFα, etc.) Luciferase NF-κB Figure 9: The principle of reporter cell lines used in the LumiMAT™ Pyrogen Detection Kit, a monocyte activation test (MAT). Trends in pyrogen testing Several factors are influencing the trends in pyrogen testing and driving innovation to find better solutions that ensure safety. The move away from animal-based tests is much needed and is driven by ethical concerns and changes in the regulatory environment. This brings impetus to the use and further development of in vitro methods like rFC assays and monocyte activation assays and provides incentives for researchers to find new ways to test for the wide range of substances classified as pyrogens. Pyrogen and endotoxin tests: Past, present and future 13 The roles of microplate readers in pyrogen testing MICROPLATE READERS AND PYROGEN TESTING LAL tests are colorimetric or turbidimetric assays. These types of assays have typically been performed on a spectrophotometer using samples in tubes or vials. However, they are also amenable to analysis using a microplate reader. rFC and MAT assays rely on either absorbance, fluorescence, or luminescence readouts and are readily carried out using a microplate reader. Microplate readers therefore offer numerous advantages for many of the available pyrogen tests and are versatile tools to assist in the quick and reliable detection of these substances when combined with commercially available, testing methods. A microplate reader is therefore a powerful gateway to the different options available for the tests and technologies used in pyrogen tests. BMG LABTECH offers a range of microplate readers with many features ideally suited for pyrogen and bacterial endotoxin tests. These include the SPECTROstar® Nano, Omega series, CLARIOstar® Plus, VANTAstar® and PHERAstar® FSX. The SPECTROstar Nano is a single-mode dedicated absorbance plate reader. As such, it can be readily used for turbidimetric and colorimetric assays including absorbance-based ELISAs for cytokine quantification upon MAT and LAL assays with colorimetric readout. All other mentioned BMG LABTECH readers have multi-mode detection capabilities, including the fluorescence and luminescence detection needed to cover assays like the recombinant factor C (rFC) and monocyte activation tests (MAT) based on a gene reporter readout. BMG LABTECH readers are equipped with an ultra-fast 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 which makes it ideal for absorbance-based ELISAs that are used for monocyte activation tests. The CLARIOstar Plus and VANTAstar additionally offer outstanding wavelength flexibility in fluorescence and Pyrogen and endotoxin tests: Past, present and future 14 luminescence with the Linear Variable Filter (LVF) MonochromatorsTM, which is an asset for many pyrogen assays. Increased light transmission and sensitivity is possible courtesy of LVF Monochromator and different filter options. The CLARIOstar Plus, VANTAstar and PHERAstar FSX include Enhanced Dynamic Range (EDR) technology for superior performance in a single luminescence or fluorescence run. This ensures accurate signal quantification across low to high concentrations of endotoxins and pyrogens in these types of assays without running into the risk of signal saturation. This feature is especially beneficial for kinetic assays where the final signal intensity is hard to predict (like the fluorescent rFC assay). BMG LABTECH microplate readers facilitate kinetic evaluations when monitoring signal changes over time. Time to threshold can be readily monitored for example in kinetic assays for turbidimetric and colorimetric LAL kits. They also permit baseline and signal increases to be detected in the same run which saves time and avoids unnecessary assays. 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 pyrogen tests. Many of the tests used have a temperature optimum at 37 °C. Only a temperature incubation option makes it possible to monitor the signal development of these assays 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. In addition, the Atmospheric Control Unit (ACU) for the CLARIOstar Plus, VANTAstar, Omega series and NEPHELOstar Plus makes it possible to maintain an optimum CO2 concentration at 5%, the ideal concentration for human cells, like the NOMO-1 cell line used in LumiMAT assays. 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. This injection option is particularly useful for luminescence-based gene reporter assays like LumiMAT as it helps to further automatise the workflow. The VANTAstar can be equipped with a modular injection unit. BMG LABTECH’s reader control software includes pre-defined protocols for the different assays and thereby offers a “Click and Start” solution for the evaluation of various kits. The MARS data analysis package is provided with all BMG LABTECH microplate readers and offers a host of features to facilitate the analysis of data from diverse pyrogen assays. Options include automated evaluations from templates suitable for different endotoxin assays, easy calculation of time to threshold, standard curve fitting and recovery rate calculations that might be used for luminescence-based MAT assays. BMG LABTECH multimode readers offer fluorescence, luminescence and absorbance detection capabilities in the same reader which means that they can readily cover the different assay options needed to test for pyrogens and endotoxins. 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. Pyrogen and endotoxin tests: Past, present and future 15 CLARIOstar® Plus VANTAstar® Suitable for: • LAL kits (colorimetric & turbidimetric) • rFC kits • ELISAs for MAT assays • LumiMAT Suitable for: • LAL kits (colorimetric & turbidimetric) • rFC kits • ELISAs for MAT assays • LumiMAT • Linear Variable Filter (LVF) Monochromators • Highly sensitive filters and an ultra-fast UV/vis spectrometer • Enhanced Dynamic Range technology • Rapid full-plate auto-focus • Endpoint and kinetic detection • Shaking (3 modes) • Up to two reagent injectors • Temperature control • Atmospheric Control Unit (ACU) • Multi-user reader control and data analysis software Absorbance Fluorescence Absorbance (incl. FRET) Fluorescence (incl. FRET) Luminescence (incl. BRET) Luminescence (incl. BRET) Up to 1536-well microplates Up to 384-well microplates Pyrogen and endotoxin tests: Past, present and future 16 SPECTROstar® Nano Omega series Suitable for: • LAL kits (colorimetric & turbidimetric) • ELISAs for MAT assays Suitable for: • LAL kits (colorimetric & turbidimetric) • rFC kits • ELISAs for MAT assays • LumiMAT • Ultra-fast UV/vis spectrometer • Integrated cuvette port • Endpoint and kinetic detection • Shaking (3 modes) • Temperature control • Multi-user reader control and data analysis software • Modular, single to multi-mode reader platform • Filters and an ultra-fast UV/vis spectrometer • Endpoint and kinetic detection • Shaking (3 modes) • Up to two reagent injectors • Temperature control • Atmospheric Control Unit (ACU) • Multi-user reader control and data analysis software Absorbance Absorbance Fluorescence (incl. FRET) Luminescence (incl. BRET) Up to 1536-well microplates Up to 384-well microplates Pyrogen and endotoxin tests: Past, present and future 17 You can learn more about pyrogens and bacterial endotoxins in the following blogs: Pyrogens and pyrogen testing | BMG LABTECH What are Endotoxins? | BMG LABTECH The LAL assay: detecting bacterial contamination | BMG LABTECH The rFC Test or Recombinant Factor C Test | BMG LABTECH The BET test or bacterial endotoxin test | BMG LABTECH The Monocyte Activation Test | BMG LABTECH NEPHELOstar® Plus Suitable for: • Turbidimetric LAL kits • Nephelometric and turbidimetric detection • Direct detection of light scattering • Endpoint and kinetic detection • Shaking (3 modes) • Up to two reagent injectors • Temperature control • Atmospheric Control Unit (ACU) • Multi-user reader control and data analysis software Up to 384-well microplates Nephelometry Turbidity Pyrogen and endotoxin tests: Past, present and future 18 A DIFFERENT ANGLE Please introduce yourself, your role at bioMérieux and your background. My name is Luca Di Bello, I have a master’s degree in biology from the Technical University of Munich in Germany. During my studies, I focused on the topics of microbiology, biochemistry and medical biology which all combine into the world of endotoxins (chemically lipopolysaccharides, LPS). I am part of the Endotoxin Application Specialist Team and based at the bioMérieux Center of Excellence for Endotoxins in Bernried, Germany. In my role, I support and develop current and new applications of the sustainable ENDONEXT™ endotoxin assays and automation platforms intended for quantitative determination of endotoxin in pharmaceutical end-products, in-process control and research samples, and medical device testing. Please briefly introduce the bioMérieux company and its main areas of focus. bioMérieux is a global diagnostics company with currently around 14 600 employees active in over 150 countries, with its headquarters in Marcy-l’Étoile, France. Our company history spans over 100 years. We develop, produce and market diagnostic and testing solutions consisting of reagents, devices, software and services for use in the medical and industrial sectors. Our advanced endotoxin detection solutions ENDONEXT™ , based on recombinant Factor C technology (rFC), were developed to meet the need for a fast, accurate, agile, and environmentally friendly testing. The ENDONEXT™ reagents do not require harvesting horseshoe crab blood, thereby reducing our customers laboratory’s environmental impact and dependence on natural resources. By adopting our rFCbased solutions, labs around the world can significantly improve their testing productivity and cost efficiency, ensure product safety, and support their sustainability goals. Furthermore, we address even the most challenging sample matrices with the different solutions in our portfolio. How are the bioMérieux endotoxin kits measured? We offer four different ENDONEXT™ rFC endotoxin assays, each tailored to specific applications, ranging from rapid processing Luca Di Bello bioMérieux Bernried, Germany Application Specialist Luca Di Bello from bioMérieux shares insights on sustainable endotoxin testing and microplate reader requirements from the perspective of a kit manufacturer Pyrogen and endotoxin tests: Past, present and future 19 with single tests, low- and high-throughput testing to handling of the most complex sample matrices. All ENDONEXT™ assays are end-point fluorescence-based microplate or microstrip assays which utilize the recombinantly produced endotoxin receptor (rFC) in combination with a fluorogenic substrate. The binding of endotoxins by rFC results in an active form of the enzyme, which in turn cleaves a fluorogenic substrate, releasing the fluorophore detectable by a reader. The resulting increase in fluorescence over time correlates with the endotoxin activity in the sample wells. Using a standard curve, all samples can be quantitatively assessed and compared against defined endotoxin release limits. With which devices can the bioMérieux kits be measured and what are the general benefits of microplate readerbased detection? The microplate reader should be capable of measuring fluorescence. Our assays are compatible with both monochromator and filter-based systems, as long as the excitation and emission wavelengths are suitable for the substrate. The recommended settings for all ENDONEXT™ assays are excitation 380/20 nm and emission 440/40 nm. Performing assays in a 96-well format significantly reduces the amount of material required per sample while greatly increasing the throughput. In addition, the workflow is much more straightforward compared to measuring each sample individually. This format enhances overall efficiency by allowing the simultaneous analysis of up to 21 samples, 1 2 3 4 5 6 7 8 9 10 11 12 STD 50 STD 50 PPC SPL 1 PPC SPL 2 PPC SPL 3 PPC SPL 4 PPC SPL 5 PPC SPL 6 PPC SPL 7 PPC SPL 8 PPC SPL 9 PPC SPL 10 STD 5 STD 5 PPC SPL 1 PPC SPL 2 PPC SPL 3 PPC SPL 4 PPC SPL 5 PPC SPL 6 PPC SPL 7 PPC SPL 8 PPC SPL 9 PPC SPL 10 STD 0,5 STD 0,5 SPL 1 SPL 2 SPL 3 SPL 4 SPL 5 SPL 6 SPL 7 SPL 8 SPL 9 SPL 10 STD 0,05 STD 0,05 SPL 1 SPL 2 SPL 3 SPL 4 SPL 5 SPL 6 SPL 7 SPL 8 SPL 9 SPL 10 STD 0,005 STD 0,005 SPL 11 SPL 12 SPL 13 SPL 14 SPL 15 SPL 16 SPL 17 SPL 18 SPL 19 SPL 20 BLK BLK SPL 11 SPL 12 SPL 13 SPL 14 SPL 15 SPL 16 SPL 17 SPL 18 SPL 19 SPL 20 BLK BLK PPC SPL 11 PPC SPL 12 PPC SPL 13 PPC SPL 14 PPC SPL 15 PPC SPL 16 PPC SPL 17 PPC SPL 18 PPC SPL 19 PPC SPL 20 PPC Ctrl PPC Ctrl PPC SPL 11 PPC SPL 12 PPC SPL 13 PPC SPL 14 PPC SPL 15 PPC SPL 16 PPC SPL 17 PPC SPL 18 PPC SPL 19 PPC SPL 20 A B C D E F G H CSE Standards Samples Negative Controls PPC Control Endotoxin rFC Fluorogenic substrate Fluorescence rFC Figure 10: Plate layout and assay principle of the ENDONEXTTM assay using the precoated GOPLATETM. ©bioMérieux SA Pyrogen and endotoxin tests: Past, present and future 20 including all necessary controls, in a single run (see fig. 10). The ENDOZYME II GO version of the kit provides users with the ready-to-use GOPLATE™ system, precoated with precise amounts of CSE for the standard curve and PPCs. Learn more about the advantages and applications of the GOPLATE™ in our Application Note 405 Streamlined detection of endotoxin using the ENDOZYME II recombinant Factor C assay and Enhanced Dynamic Range on the VANTAstar. Ultimately, these kits facilitate scalability, making it easier to automate and upscale the process for highthroughput environments. What are the advantages of a Fluorescence-based readout? Compared to traditional absorbance-based methods, fluorescence detection offers significantly higher sensitivity. This enhanced sensitivity makes it particularly well-suited for the endotoxin detection with rFC, which requires a precise quantification especially across very low signal levels. A further important advantage of endotoxin detection using fluorescence is that coloured and turbid samples are less likely to interfere with the measurement, as opposed to traditional LAL methods using absorbance, where interference due to colour and/or turbidity is a widely acknowledged problem. From a kit manufacturer perspective, what features should customers pay particular attention to when purchasing microplate readers for use with bioMérieux kits? The minimum requirement for a microplate reader is the possibility to measure fluorescence with the correct wavelengths. It should also feature an integrated incubation function, allowing the plate to be maintained at 37 °C directly within the reader. An internal shaking function further simplifies the workflow of mixing the assay reagents with the sample wells. A high-quality reader with strong sensitivity and low intrinsic variability will have a positive impact on assay results. For many customers, the option of a multi-mode reader can be a significant advantage. It allows for the measurement of both traditional absorbance-based endotoxin detection methods and modern fluorescencebased assays, making the transition between technologies seamless and uncomplicated. If it is the need, the reader should be capable also of performing kinetic measurements. The software must be capable of performing at least a linear regression with logarithmic transformation to generate standard curves with a maximum concentration of 5 EU/mL. For applications requiring quantification up to 50 EU/mL, polynomial regression functionality is essential. Furthermore, in a pharmaceutical quality control (QC) environment, it is critical that the software complies with 21 CFR Part 11 regulations to ensure data integrity and regulatory adherence. What is your experience with ENDONEXT kits on BMG LABTECH microplate readers? In collaboration with BMG Labtech, an application note was published showcasing the successful implementation of the ENDOZYME® II assay on the VANTAstar® microplate reader by the endotoxin testing unit of medical device-manufacturer CODAN Medizinische Geräte GmbH. A key feature highlighted in this context was the reader’s Enhanced Dynamic Range (EDR) setting, which significantly enhances user convenience when working with fluorescence-based assay kits. By eliminating the need for manual gain adjustment, the EDR mode streamlines the workflow and ensures consistent and high-quality results. Furthermore, BMG Labtech offered tailored support to the testing laboratory with MARS Data Analysis Software master templates for ENDOZYME® II according to their specific needs in terms of both kinetic reading mode, assay validity criteria, and reporting compliant with 21 CFR Part 11. Pyrogen and endotoxin tests: Past, present and future 21 Industry-wide applications for pyrogen tests CODA New innovations for pyrogen tests that deliver improved safety and scale will bring further savings in costs and time and will benefit from the automation friendly approaches offered by microplate readers. Traditional pyrogen testing methods often require labor-intensive manual preparation leading to inefficiencies, increased costs, and inaccurate results. The availability of new tests and further refinements to pyrogen tests are reducing inefficiencies by implementing automation-compatible testing methods, decreasing costs through the implementation of sustainable practices, and improving testing performance by generating improved sensitivity of testing methods. In methods like the monocyte activation test, workflows have been reduced from days to hours with appropriate quality control. Modifications to existing protocols are extending applications to a wider range of interventions, for example by allowing direct detection of pyrogens on the surfaces of a wider range of medical devices. As highlighted in this eBook, some of these types of advances are already reaching the life sciences. Instead of elaborate multi-step processes like incubating cells with samples and using the supernatant for immunoassays, assays like the LumiMAT assay deliver an addmix- read approach that significantly reduces handling steps and simplifies the transfer to automated systems. The availability of tests like the ENDOZYME II GO kit, a ready-to-go microplate precoated with standard and positive controls, saves time and eliminates dilution and reconstitution steps. These developments are already paving the way for higher throughput pyrogen tests. Regulation standards will continue to advance which will require further innovation and provide impetus for improved tests for pyrogens. As such, pyrogen tests will be relevant and applicable to the increasing number of products arising from biotechnology, drug discovery, microbiology, immunology and many other areas of quality control and bioanalysis. Pyrogen and endotoxin tests: Past, present and future 22 REFERENCES 1. Pyrogens, Still a Danger | FDA https://www.fda.gov/inspectionscompliance- enforcement-and-criminalinvestigations/ inspection-technical-guides/ pyrogens-still-danger. Accessed 03/20/2025 2. Material-mediated pyrogenicity. Prakash Srinivasan Timiri Shanmugam, Thamizharasan Sampath, Indumathy Jagadeeswaran, Sandhiya Thamizharasan, Safura Fathima, Editor(s): Prakash Srinivasan Timiri Shanmugam, Thamizharasan Sampath, Indumathy Jagadeeswaran, Biocompatibility Protocols for Medical Devices and Materials, Academic Press, 2023, Pages 55-66, ISBN 9780323919524, https://doi.org/10.1016/ B978-0-323-91952-4.00009-X. 3. 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. 4. Schneier M, Razdan S, Miller AM, Briceno ME, Barua S. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Biotechnol Bioeng. 2020 Aug;117(8):2588-2609. doi: 10.1002/bit.27362. 5. Schwadner, R. et al. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. J. Biol. Chem. 274, 17406 17409 (1999). 6. Hort, E,. Penfold, W.J., A Critical Study of Experimental Fever. Lister Institute of Preventive Medicine, March 14, 1912. 7. 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. 8. Bang FB. The toxic effect of a marine bacterium on Limulus and the formation of blood clots. Biol. Bull. (1953) 105:447-448. 9. Bang FB. A bacterial disease of Limulus polyphemus. Bull. Johns Hopkins Hosp. (1956) 98:325. 10. Levin J and Bang FB. A description of cellular coagulation in Limulus. Bull. Johns Hopkins Hosp. (1964) 115:337. 11. Levin J and Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. (1964) 115:265.6. 12. Levin J, Bang FB. Clottable protein in Limulus: Its localization and kinetics of its coagulation by endotoxin. Thromb. Diathes. Haemorrh. (Stuttg) (1968) 19:186. 13. Maloney T, Phelan R, Simmons N. Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLoS Biol. 2018 Oct 12;16(10):e2006607. doi: 10.1371/journal. pbio.2006607. 14. 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. 15. Bolden J, Smith K. Application of Recombinant Factor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products. PDA J. Pharm. Sci. Technol. 2017 71(5):405-412. doi: 10.5731/pdajpst.2017.007849. 16. Brown J, Clippinger AJ, Fritz Briglia C, Casey W, Coleman K, Fritsch A, Hartung T, Maouyo D, Muller T, Reich J, Robert L, Roeder R, Sanchez G, Sawyer AY, Solati S, Tirumalai R, Zwisler W, Allen D. Using the monocyte activation test as a stand-alone release test for medical devices. ALTEX. 2021;38(1):151- 156. doi: 10.14573/altex.2012021. 17. Hartung T. Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test. ALTEX. 2021;38(1):3-19. doi: 10.14573/altex.2101051. 18. European Pharmacopoeia to put an end to the rabbit pyrogen test - European Directorate for the Quality of Medicines & HealthCare, press release European Directorate for the Quality of Medicines & Healthcare, accessed 8 May 2025. Pyrogen and endotoxin tests: Past, present and future 23 APPLICATION NOTES Microplate-based assays offer significant benefits for pyrogen and endotoxin tests. In this section, we have compiled some of the application notes available from BMG LABTECH that were developed with the help of our customers and kit manufacturers, and which are relevant to the testing of pyrogens and endotoxins. BMG LABTECH’s application notes reflect work performed in partnership with our customers on our instruments. They offer insight into specific assays and applications and demonstrate what is possible using BMG LABTECH microplate readers. The application notes in this section cover the main types of pyrogen tests that can be performed in a microplate. They demonstrate applications that involve absorbance, fluorescence and luminescence measurements. The examples span in vitro biochemical assays to cell-based assays and demonstrate not only the scope of what is possible but also some of the resources needed to implement these pyrogen tests. Pyrogen tests are vital to ensure the safety of different health interventions including pharmaceuticals, biological therapeutics, medical devices and an array of other products. Over time, we will add more examples on pyrogen testing to our application note portfolio on our web site at https://www.bmglabtech.com/ en/application-notes. Please check-in for the latest updates. You are also welcome to contact us at [email protected] if you are interested in working with us on a future application note. Pyrogen and endotoxin tests: Past, present and future 24 Application notes in this section Bacterial endotoxin test • AN237: Endotoxin detection on a microplate reader using a colorimetric, kinetic endotoxin detection kit with integrated data analysis • AN409: Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay • AN412: Colorimetric and turbidimetric analysis of endotoxins using the absorbance detection mode Recombinant factor C • AN343: Faster PyroGene™ detection of endotoxin using Enhanced Dynamic Range on the CLARIOstar Plus • AN405: Stream-lined detection of endotoxin using the ENDOZYME II recombinant Factor C assay and Enhanced Dynamic Range on the VANTAstar Monocyte activation test • AN408: Fast and highly sensitive pyrogen detection with the LumiMAT™ assay performed on BMG LABTECH microplate readers Others • AN184: Multiplex analysis of inflammatory cytokines from primary human macrophages using a FLUOstar Omega • AN341: Highly sensitive ELISAs in 90-minutes: SimpleStep ELISA® kits and the SPECTROstar® Nano Here you will find a list of all the protocol files for import into your BMG LABTECH control software and the corresponding templates for automatic evaluation of generated data in MARS for all application notes mentioned in this eBook. You can download these from the overview page, import them into your BMG LABTECH software and use them to evaluate your analysis. Pyrogen and endotoxin tests: Past, present and future 25 O O O O O NH NH O O O O O O O O O O O O O O O O P P O O OH OH OH OH OH HO HO HO HO HO HO HO OH HO HO OH Endotoxin detection on a microplate reader using a colorimetric, kinetic endotoxin detection kit with integrated data analysis Fig. 1: Structure of the (3-deoxy-D-manno-octulosonic acid) 2 Lipid A endotoxin from E. coli K-12. Figure licensed under the Creative Commons Attribution-Share Alike 3.0 Unported licenses and adapted from: http://www.jlr.org/content/47/5/1097.full. For endotoxin quantifi cation, Lonza has developed a kinetic endotoxin detection kit, which utilizes the coagulation properties of horseshoe crab blood in the presence of even low levels of endotoxin. The clottable protein was isolated and is the active component of the Limulus Amebocyte Lysate (LAL) in the endotoxin detection kit. In the presence of endotoxin, a proenzyme in the LAL is activated which cleaves a colorless peptide, Ac-Ile-Glu-Ala-Arg-pNA, releasing p-nitroaniline (pNA) which can be detected by continuous absorbance measurements at 405 nm. Endotoxin detection is achieved and the concentration of endotoxin is calculated by comparing reaction times of samples to solutions of known amounts of endotoxin. The reaction time is typically defi ned as the time it takes to produce a 0.2 OD change in absorbance at 405 nm. The endotoxin concentration is inversely proportional to the reaction time so a smaller reaction time indicates a higher endotoxin concentration. The microplate reader measures the kinetic endotoxin detection kit readily and in conjunction with MARS data analysis software can produce reaction times. MARS can also plot the standards and interpolate unknown samples from a linear regression or polynomial fi t. Proenzyme Endotoxin Enzyme Enzyme Substrate + H2O Peptide + pNA MATERIALS & METHODS ∙ Corning 96 well Microplate, Clear ∙ Lonza Kinetic-QCLTM endotoxin detection kit ∙ Filter-based or spectrometer equipped microplate reader from BMG LABTECH 100 μl of standards and unknowns were measured with the detection reagent for absorption at 405 nm in duplicate for a total of 100 min in plate mode (slow kinetics). After reagent addition a reading was taken every 2.5 min for a total of 40 points. Standards included 0.005, 0.05, 0.5 and 5.0 EU/ml, where EU = endotoxic units, a comparative measure of endotoxin activity. Since a baseline correction is applied, blanks are not required for optimal assay performance when using a kinetic endotoxin detection kit. Instrument settings Detection Mode: Absorbance, plate mode kinetic Optics: 405 nm No. of cycles: 40 Cycle Time: 150 sec Chris Quinlan and Carl Peters, BMG LABTECH To avoid unwanted infl ammatory responses it is important that DNA samples are endotoxin free The Lonza endotoxin detection kit was performed on a fi lter-based microplate reader Use of MARS Data Analysis simplifi es data interpretation of the endotoxin detection kit INTRODUCTION Endotoxins or lipopolysaccharides (LPS) are undesirable byproducts of gram-negative bacterial preparations often found in plasmid DNA and ovalbumin preps. LPS is located in the outer membrane of the bacterial cell. Even trace amounts cause a signifi cant infl ammatory response. The presence of endotoxin in the blood (endotoxemia) can lead to sepsis in mammals. Therefore, endotoxin detection is essential to ensure elimination of endotoxin from DNA and protein preparations to avoid unwanted responses in in vivo and in vitro assays ASSAY PRINCIPLE NEPH AS FP TRF & TR-FRET LUM & BRET FI & FRET ABS AN 237 SPECTROstar Nano Omega series VANTAstar CLARIOstar Plus PHERAstar FSX O O O O O NH NH O O O O O O O O O O O O O O O O P P O O OH OH OH OH OH HO HO HO HO HO HO HO OH HO HO OH Endotoxin detection on a microplate reader using a colorimetric, kinetic endotoxin detection kit with integrated data analysis Fig. 1: Structure of the (3-deoxy-D-manno-octulosonic acid) 2 Lipid A endotoxin from E. coli K-12. Figure licensed under the Creative Commons Attribution-Share Alike 3.0 Unported licenses and adapted from: http://www.jlr.org/content/47/5/1097.full. For endotoxin quantifi cation, Lonza has developed a kinetic endotoxin detection kit, which utilizes the coagulation properties of horseshoe crab blood in the presence of even low levels of endotoxin. The clottable protein was isolated and is the active component of the Limulus Amebocyte Lysate (LAL) in the endotoxin detection kit. In the presence of endotoxin, a proenzyme in the LAL is activated which cleaves a colorless peptide, Ac-Ile-Glu-Ala-Arg-pNA, releasing p-nitroaniline (pNA) which can be detected by continuous absorbance measurements at 405 nm. Endotoxin detection is achieved and the concentration of endotoxin is calculated by comparing reaction times of samples to solutions of known amounts of endotoxin. The reaction time is typically defi ned as the time it takes to produce a 0.2 OD change in absorbance at 405 nm. The endotoxin concentration is inversely proportional to the reaction time so a smaller reaction time indicates a higher endotoxin concentration. The microplate reader measures the kinetic endotoxin detection kit readily and in conjunction with MARS data analysis software can produce reaction times. MARS can also plot the standards and interpolate unknown samples from a linear regression or polynomial fi t. Proenzyme Endotoxin Enzyme Enzyme Substrate + H2O Peptide + pNA MATERIALS & METHODS ∙ Corning 96 well Microplate, Clear ∙ Lonza Kinetic-QCLTM endotoxin detection kit ∙ Filter-based or spectrometer equipped microplate reader from BMG LABTECH 100 μl of standards and unknowns were measured with the detection reagent for absorption at 405 nm in duplicate for a total of 100 min in plate mode (slow kinetics). After reagent addition a reading was taken every 2.5 min for a total of 40 points. Standards included 0.005, 0.05, 0.5 and 5.0 EU/ml, where EU = endotoxic units, a comparative measure of endotoxin activity. Since a baseline correction is applied, blanks are not required for optimal assay performance when using a kinetic endotoxin detection kit. Instrument settings Detection Mode: Absorbance, plate mode kinetic Optics: 405 nm No. of cycles: 40 Cycle Time: 150 sec Chris Quinlan and Carl Peters, BMG LABTECH To avoid unwanted infl ammatory responses it is important that DNA samples are endotoxin free The Lonza endotoxin detection kit was performed on a fi lter-based microplate reader Use of MARS Data Analysis simplifi es data interpretation of the endotoxin detection kit INTRODUCTION Endotoxins or lipopolysaccharides (LPS) are undesirable byproducts of gram-negative bacterial preparations often found in plasmid DNA and ovalbumin preps. LPS is located in the outer membrane of the bacterial cell. Even trace amounts cause a signifi cant infl ammatory response. The presence of endotoxin in the blood (endotoxemia) can lead to sepsis in mammals. Therefore, endotoxin detection is essential to ensure elimination of endotoxin from DNA and protein preparations to avoid unwanted responses in in vivo and in vitro assays ASSAY PRINCIPLE NEPH AS FP TRF & TR-FRET LUM & BRET FI & FRET ABS AN 237 SPECTROstar Nano Omega series VANTAstar CLARIOstar Plus PHERAstar FSX Pyrogen and endotoxin tests: Past, present and future 26 OD 2.6 2.2 1.8 1.4 1 0.6 0.2 0 8 16 24 32 40 48 56 64 72 80 88 96 Time in minutes Fig. 2: Signal curves for several endotoxin samples obtained with the endotoxin detection kit. Figure is directly taken from the MARS data analysis software. The kinetic data of the endotoxin detection kit was evaluated utilizing the MARS data analysis software from BMG LABTECH. A baseline correction was applied to the raw data by subtracting cycle 1. The reaction time was calculated by performing a “time to threshold calculation” on the baseline corrected raw data generated with the endotoxin detection kit. The threshold value was set for 0.2 OD yielding a reaction time in seconds. The average reaction time was plotted against concentration in EU/ml using a linear regression model (Fig. 3). Reaction time (min) 00:16:40 0.01 0.1 1 [Endotoxin], EU/ml Fig. 3: Linear Regression Fit of Standards (log/log). The resulting standard curve fi t had an R2 value of 0.999 allowing for reliable interpolation of unknown samples (Table 1). Sample [Endotoxin], EU/ml calculated from Linear Regression Fit Unknown 1 4.59 Unknown 2 0.38 Alternatively, the MARS data analysis software facilitated the plotting of standards utilizing a polynomial curve fi t. It is recommended that the polynomial order be one less than the number of standards used. For this endotoxin detection kit, a 3rd order polynomial fi t could be used. Standard Curve Fit Select the input data: Time To Threshold Linear regression fit Select the calculation method 4-Parameter fit 5-Parameter fit Cubic spline fit Point to point fit Segmental regression fit 2nd Polynomial fit 3rd Polynomial fit Hyperbola fit Linear regression fit 4-Parameter fit 5-Parameter fit Cubic spline fit Point to point fit Segmental regression fit 2nd Polynomial fit 3rd Polynomial fit Hyperbola fit Result display name: X values linear logarithmic Y values linear logarithmic Linear regression fit Fig. 4: Overview of standard curve fi ts in the MARS data analysis software. CONCLUSION The combination of sensitive microplate readers and the powerful MARS data analysis package allows for easy handling of complex assays such as the Kinetic-QCL endotoxin detection kit from Lonza. The MARS data analysis package is standard with all BMG LABTECH readers and c an be installed on other computers in the same lab at no additional charge. This endotoxin detection kit can also be measured by all BMG LABTECH microplate readers that measure absorbance including the SPECTROstar® Nano, PHERAstar® FSX, FLUOstar® Omega, VANTAstar® and The MARS data analysis software further allows for the creation of a template for the analysis of the endotoxin detection kit and other sophisticated calculations (Fig. 4) to provide immediate data reduction and curve fi ts as soon as the assay is completed. Review 07/2013 Keywords: Endotoxin, Horseshoe crab, LAL assay, Limulus amebocyte, Lipopolysaccharide RESULTS & DISCUSSION The absorbance measurements over time resulted in different signal curves (fi g. 2). OD 2.6 2.2 1.8 1.4 1 0.6 0.2 0 8 16 24 32 40 48 56 64 72 80 88 96 Time in minutes Fig. 2: Signal curves for several endotoxin samples obtained with the endotoxin detection kit. Figure is directly taken from the MARS data analysis software. The kinetic data of the endotoxin detection kit was evaluated utilizing the MARS data analysis software from BMG LABTECH. A baseline correction was applied to the raw data by subtracting cycle 1. The reaction time was calculated by performing a “time to threshold calculation” on the baseline corrected raw data generated with the endotoxin detection kit. The threshold value was set for 0.2 OD yielding a reaction time in seconds. The average reaction time was plotted against concentration in EU/ml using a linear regression model (Fig. 3). Reaction time (min) 00:16:40 0.01 0.1 1 [Endotoxin], EU/ml Fig. 3: Linear Regression Fit of Standards (log/log). The resulting standard curve fi t had an R2 value of 0.999 allowing for reliable interpolation of unknown samples (Table 1). Sample [Endotoxin], EU/ml calculated from Linear Regression Fit Unknown 1 4.59 Unknown 2 0.38 Alternatively, the MARS data analysis software facilitated the plotting of standards utilizing a polynomial curve fi t. It is recommended that the polynomial order be one less than the number of standards used. For this endotoxin detection kit, a 3rd order polynomial fi t could be used. Standard Curve Fit Select the input data: Time To Threshold Linear regression fit Select the calculation method 4-Parameter fit 5-Parameter fit Cubic spline fit Point to point fit Segmental regression fit 2nd Polynomial fit 3rd Polynomial fit Hyperbola fit Linear regression fit 4-Parameter fit 5-Parameter fit Cubic spline fit Point to point fit Segmental regression fit 2nd Polynomial fit 3rd Polynomial fit Hyperbola fit Result display name: X values linear logarithmic Y values linear logarithmic Linear regression fit Fig. 4: Overview of standard curve fi ts in the MARS data analysis software. CONCLUSION The combination of sensitive microplate readers and the powerful MARS data analysis package allows for easy handling of complex assays such as the Kinetic-QCL endotoxin detection kit from Lonza. The MARS data analysis package is standard with all BMG LABTECH readers and c an be installed on other computers in the same lab at no additional charge. This endotoxin detection kit can also be measured by all BMG LABTECH microplate readers that measure absorbance including the SPECTROstar® Nano, PHERAstar® FSX, FLUOstar® Omega, VANTAstar® and The MARS data analysis software further allows for the creation of a template for the analysis of the endotoxin detection kit and other sophisticated calculations (Fig. 4) to provide immediate data reduction and curve fi ts as soon as the assay is completed. Review 07/2013 Keywords: Endotoxin, Horseshoe crab, LAL assay, Limulus amebocyte, Lipopolysaccharide RESULTS & DISCUSSION The absorbance measurements over time resulted in different signal curves (fi g. 2). Pyrogen and endotoxin tests: Past, present and future 27 +Endotoxin Light Source Absorbance Detector Nephelometry Detector Adding endotoxin starts clotting reaction Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay Martin Mangold BMG LABTECH, Ortenberg, Germany INTRODUCTION Bacterial endotoxins, also known as lipopolysaccharides (LPS), are potent pyrogens found in the outer membrane of Gramnegative bacteria.1,2 Their presence in pharmaceuticals, medical devices, and biological products can trigger severe infl ammatory reactions in our immune system. These infl ammatory reactions may lead to serious adverse events and compromise health. Therefore, the detection and quantifi cation of endotoxins are crucial for ensuring product safety and regulatory compliance for food, medical, and other products. The Limulus Amebocyte Lysate (LAL) assay is a widely used method for detecting bacterial endotoxins.3-5 Two types of horseshoe crab have been used as sources of LAL: Limulus polyphemus from the North Atlantic and Tachypleus spp. from Asia. The LAL assay exploits the natural clotting response of blood cells (amebocytes) isolated from these animals to endotoxins. When exposed to endotoxins, LAL undergoes a series of enzymatic reactions that result in gel formation, which can be measured to determine the presence and concentration of endotoxins. The PYROSTAR ES-F/Plate LAL test is sensitive, robust, suitable for small sample volumes, and is not activated by β-glucans which can lead to false positive results. It therefore ensures that products are free from harmful levels of endotoxins, safeguarding patient health and meeting stringent regulatory standards. ASSAY PRINCIPLE The PYROSTAR ES-F/Plate kit is based on the classic LAL assay principle. In the presence of endotoxins, the LAL responds with a clotting reaction. Clotted LAL can be detected on a microplate reader in two ways which both depend on light scattering but use different types of readouts. The LAL assay can either be performed with a light-scattering readout on a nephelometry-based microplate reader or with an turbidimetric readout on a spectrometer-based absorbance microplate reader (Figure 1). In a nephelometer, scattered light is measured due to detection taking place at an angle to the incident light. If an absorbance-based microplate reader is used, light that passes through the sample is measured - so all light that isn’t scattered. A reference measurement is used to determine the total light produced by the spectrometer, which allows an indirect statement to be made about the amount of light scattered by the sample. MATERIALS & METHODS ∙ 96-well plates, clear, polystyrene (Greiner, 655101) ∙ PYROSTAR ES-F/Plate kit (Wako, 543-10331) ∙ Endotoxin-free water ∙ NEPHELOstar Plus (BMG LABTECH) ∙ VANTAstar (BMG LABTECH) Experimental Procedure 1,000 EU/mL control standard endotoxin stock was prepared in endotoxin-free water. Using the stock solution, a 10-fold dilution series was prepared with endotoxin-free water according to the kit protocol (0.001 to 100 EU/mL). A vial of PYROSTAR ES-F reagent containing the LAL was dissolved in 2 mL of endotoxin-free water. 50 μL of the endotoxin dilution series was pipetted into a clear 96-well microplate. The detection reaction was started by adding 50 μL of PYROSTAR ES-F reagent and the plate was immediately read on the NEPHELOstar Plus or VANTAstar for 60 min at 37°C. Instrument settings Fig. 1: PYROSTAR ES-F/Plate LAL assay principle Limulus amebocyte lysate (LAL)-based approach for the detection of trace amounts of endotoxin based on light scattering Light scattering assays can be analysed using a microplate reader with either nephelometric or turbidimetric readouts The PYROSTAR™ ES-F/Plate LAL assay can be performed on the NEPHELOstar® Plus or spectrometer- based readers such as the VANTAstar® NEPH AS FP TRF & TR-FRET LUM & BRET FI & FRET ABS AN 409 NEPHELOstar Plus SPECTROstar Nano Omega series VANTAstar CLARIOstar Plus PHERAstar FSX Optic settings Nephelometry, plate mode Laser intensity 80 % Kinetic settings Number of cycles 90 Cycle time 40 s Incubation Target Temperature 37 °C Optic settings Absorbance, plate mode Spectrometer 405 nm General settings Flashes 22 Kinetic settings Number of cycles 90 Cycle time 40 s Incubation Target Temperature 37 °C +Endotoxin Light Source Absorbance Detector Nephelometry Detector Adding endotoxin starts clotting reaction Detection of bacterial endotoxins using the PYROSTAR™ ES-F/Plate LAL assay Martin Mangold BMG LABTECH, Ortenberg, Germany INTRODUCTION Bacterial endotoxins, also known as lipopolysaccharides (LPS), are potent pyrogens found in the outer membrane of Gramnegative bacteria.1,2 Their presence in pharmaceuticals, medical devices, and biological products can trigger severe infl ammatory reactions in our immune system. These infl ammatory reactions may lead to serious adverse events and compromise health. Therefore, the detection and quantifi cation of endotoxins are crucial for ensuring product safety and regulatory compliance for food, medical, and other products. The Limulus Amebocyte Lysate (LAL) assay is a widely used method for detecting bacterial endotoxins.3-5 Two types of horseshoe crab have been used as sources of LAL: Limulus polyphemus from the North Atlantic and Tachypleus spp. from Asia. The LAL assay exploits the natural clotting response of blood cells (amebocytes) isolated from these animals to endotoxins. When exposed to endotoxins, LAL undergoes a series of enzymatic reactions that result in gel formation, which can be measured to determine the presence and concentration of endotoxins. The PYROSTAR ES-F/Plate LAL test is sensitive, robust, suitable for small sample volumes, and is not activated by β-glucans which can lead to false positive results. It therefore ensures that products are free from harmful levels of endotoxins, safeguarding patient health and meeting stringent regulatory standards. ASSAY PRINCIPLE The PYROSTAR ES-F/Plate kit is based on the classic LAL assay principle. In the presence of endotoxins, the LAL responds with a clotting reaction. Clotted LAL can be detected on a microplate reader in two ways which both depend on light scattering but use different types of readouts. The LAL assay can either be performed with a light-scattering readout on a nephelometry-based microplate reader or with an turbidimetric readout on a spectrometer-based absorbance microplate reader (Figure 1). In a nephelometer, scattered light is measured due to detection taking place at an angle to the incident light. If an absorbance-based microplate reader is used, light that passes through the sample is measured - so all light that isn’t scattered. A reference measurement is used to determine the total light produced by the spectrometer, which allows an indirect statement to be made about the amount of light scattered by the sample. MATERIALS & METHODS ∙ 96-well plates, clear, polystyrene (Greiner, 655101) ∙ PYROSTAR ES-F/Plate kit (Wako, 543-10331) ∙ Endotoxin-free water ∙ NEPHELOstar Plus (BMG LABTECH) ∙ VANTAstar (BMG LABTECH) Experimental Procedure 1,000 EU/mL control standard endotoxin stock was prepared in endotoxin-free water. Using the stock solution, a 10-fold dilution series was prepared with endotoxin-free water according to the kit protocol (0.001 to 100 EU/mL). A vial of PYROSTAR ES-F reagent containing the LAL was dissolved in 2 mL of endotoxin-free water. 50 μL of the endotoxin dilution series was pipetted into a clear 96-well microplate. The detection reaction was started by adding 50 μL of PYROSTAR ES-F reagent and the plate was immediately read on the NEPHELOstar Plus or VANTAstar for 60 min at 37°C. Instrument settings Fig. 1: PYROSTAR ES-F/Plate LAL assay principle Limulus amebocyte lysate (LAL)-based approach for the detection of trace amounts of endotoxin based on light scattering Light scattering assays can be analysed using a microplate reader with either nephelometric or turbidimetric readouts The PYROSTAR™ ES-F/Plate LAL assay can be performed on the NEPHELOstar® Plus or spectrometer- based readers such as the VANTAstar® NEPH AS FP TRF & TR-FRET LUM & BRET FI & FRET ABS AN 409 NEPHELOstar Plus SPECTROstar Nano Omega series VANTAstar CLARIOstar Plus PHERAstar FSX Optic settings Nephelometry, plate mode Laser intensity 80 % Kinetic settings Number of cycles 90 Cycle time 40 s Incubation Target Temperature 37 °C Optic settings Absorbance, plate mode Spectrometer 405 nm General settings Flashes 22 Kinetic settings Number of cycles 90 Cycle time 40 s Incubation Target Temperature 37 °C Pyrogen and endotoxin tests: Past, present and future 28 Review 08/2025 Keywords: Endotoxin, LAL assay, limulus amebocyte, lipopolysaccharide, quality control RESULTS & DISCUSSION Figure 2 shows kinetic data of the PYROSTAR ES-F/Plate kit. Samples containing endotoxin displayed an increase in assay signal over time. Higher endotoxin concentrations led to a faster increase in measurement signal until the signals reached a maximum compared to samples with lower endotoxin concentrations. The nephelometric readout (fi gure 2A) and turbidimetric readout (fi gure 2B) gave comparable results regarding to the separation of endotoxin standards. Endotoxin standard samples were used to prepare a standard curve for the LAL assay. For this purpose, fi rst time-to-threshold calculations were performed in the MARS data analysis software. For the nephelometric readout the threshold value was set to 5% of the maximum signal, while a threshold of 0.015 OD was set for the turbidimetric LAL assay. Time-tothreshold values of the endotoxin standards were then fi tted against the standard concentrations using a logarithmic linear regression fi t (fi gure 3). CONCLUSION The PYROSTAR ES-F/Plate LAL assay could be performed with both detection modes. Both the nephelometric and the turbidimetric readout yielded comparable results with the same number of endotoxin standards, although the use of the laser-equipped NEPHELOstar Plus slightly improved overall data quality. In the end both readers, absorbance- and nephelometry- based, were well suited for measuring the PYROSTAR ES-F/Plate LAL assay. REFERENCES 1. Pyrogens, Still a Danger | FDA https://www.fda.gov/inspections-compliance-enforcementand- criminal-investigations/inspection-technical-guides/ pyrogens-still-danger. Accessed 03/20/2025 2. 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. 3. Levin J and Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. (1964) 115:265.6 4. United States Pharmacopeial Convention. Committee of Revision. “The United States Pharmacopeia.” United States Pharmacopeial Convention, Incorporated, 1985. www.usp.org 5. European Pharmacopoeia Commission, and European Directorate for the Quality of Medicines & Healthcare. European pharmacopoeia. Council of Europe. www.edqm.eu/en/european-pharmacopoeia Time in minutes 760.000 720.000 680.000 640.000 600.000 560.000 520.000 480.000 440.000 400.000 360.000 320.000 280.000 240.000 200.000 160.000 120.000 80.000 40.000 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Time in minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Delta in RNU 100 EU/mL 10 EU/mL 1 EU/mL 0.1 EU/mL A B 0,160 0,150 0,140 0,130 0,120 0,110 0,100 0,090 0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 -8,97EA OD 0.01 EU/mL 0.001 EU/mL 1000 0,010 0,100 1,000 10,000 100,000 Endotoxin concentration in EU/mL Time to threshold in s Fig. 2: Kinetic readout of standard samples of the PYROSTAR ES-F/Plate kit. (A) Nephelometric readout on the NEPHELOstar Plus. (B) Turbidimetric readout on the VANTAstar microplate reader using the absorbance detection mode. Standard curves are derived from the mean of three replicates. Fig. 3: Standard curve based on nephelometric PYROSTAR ES-F/Plate LAL assay data. Time-to-threshold values were calculated from the kinetic test run and fi tted against standard concentrations using a logarithmic linear regression fi t. Review 08/2025 Keywords: Endotoxin, LAL assay, limulus amebocyte, lipopolysaccharide, quality control RESULTS & DISCUSSION Figure 2 shows kinetic data of the PYROSTAR ES-F/Plate kit. Samples containing endotoxin displayed an increase in assay signal over time. Higher endotoxin concentrations led to a faster increase in measurement signal until the signals reached a maximum compared to samples with lower endotoxin concentrations. The nephelometric readout (fi gure 2A) and turbidimetric readout (fi gure 2B) gave comparable results regarding to the separation of endotoxin standards. Endotoxin standard samples were used to prepare a standard curve for the LAL assay. For this purpose, fi rst time-to-threshold calculations were performed in the MARS data analysis software. For the nephelometric readout the threshold value was set to 5% of the maximum signal, while a threshold of 0.015 OD was set for the turbidimetric LAL assay. Time-tothreshold values of the endotoxin standards were then fi tted against the standard concentrations using a logarithmic linear regression fi t (fi gure 3). CONCLUSION The PYROSTAR ES-F/Plate LAL assay could be performed with both detection modes. Both the nephelometric and the turbidimetric readout yielded comparable results with the same number of endotoxin standards, although the use of the laser-equipped NEPHELOstar Plus slightly improved overall data quality. In the end both readers, absorbance- and nephelometry- based, were well suited for measuring the PYROSTAR ES-F/Plate LAL assay. REFERENCES 1. Pyrogens, Still a Danger | FDA https://www.fda.gov/inspections-compliance-enforcementand- criminal-investigations/inspection-technical-guides/ pyrogens-still-danger. Accessed 03/20/2025 2. 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. 3. Levin J and Bang FB. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopkins Hosp. (1964) 115:265.6 4. United States Pharmacopeial Convention. Committee of Revision. “The United States Pharmacopeia.” United States Pharmacopeial Convention, Incorporated, 1985. www.usp.org 5. European Pharmacopoeia Commission, and European Directorate for the Quality of Medicines & Healthcare. European pharmacopoeia. Council of Europe. www.edqm.eu/en/european-pharmacopoeia Time in minutes 760.000 720.000 680.000 640.000 600.000 560.000 520.000 480.000 440.000 400.000 360.000 320.000 280.000 240.000 200.000 160.000 120.000 80.000 40.000 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Time in minutes 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 Delta in RNU 100 EU/mL 10 EU/mL 1 EU/mL 0.1 EU/mL A B 0,160 0,150 0,140 0,130 0,120 0,110 0,100 0,090 0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 -8,97EA OD 0.01 EU/mL 0.001 EU/mL 1000 0,010 0,100 1,000 10,000 100,000 Endotoxin concentration in EU/mL Time to threshold in s Fig. 2: Kinetic readout of standard samples of the PYROSTAR ES-F/Plate kit. (A) Nephelometric readout on the NEPHELOstar Plus. (B) Turbidimetric readout on the VANTAstar microplate reader using the absorbance detection mode. Standard curves are derived from the mean of three replicates. Fig. 3: Standard curve based on nephelometric PYROSTAR ES-F/Plate LAL assay data. Time-to-threshold values were calculated from the kinetic test run and fi tted against standard concentrations using a logarithmic linear regression fi t. Pyrogen and endotoxin tests: Past, present and future 29 +Endotoxin Light Source Absorbance Detector High light transmission Lower light transmission Light Source Absorbance Detector Colorimetric and turbidimet