PHERAstar FSX
Powerful and most sensitive HTS plate reader
Introduction
Pyrogens are a broad range of substances that can result in adverse effects in the body due to increases in body temperature.1,2 In many cases they can trigger fever in humans and other animals due to their impact on the innate immune system. Pyrogen testing is therefore a much-needed quality control step to ensure the safety of any substance, material or device that might contain or lead to the presence of unwanted pyrogens in the body. Several methods are available to test pyrogens and endotoxins, a specific and widely encountered type of pyrogen found in the outer membrane of Gram-negative bacteria (Fig. 1). 3,4 Microplate readers are useful tools to perform many of these crucial assays that help to ensure health and safety through quality control and bioanalysis.
In this blog, we consider pyrogens and their effects, provide an overview of developments in pyrogen testing, and explore how microplate readers can help perform pyrogen tests.
What are pyrogens?
Pyrogens are a broad range of substances that can produce a rise 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 parenteral administration. Parenteral administration refers to the introduction of substances into the body by routes other than the gastrointestinal system (oral route) and typically but not exclusively involves injection or infusion directly into the bloodstream. 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 fever and serious adverse events.
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).
A further useful distinction for the diverse substances that make up pyrogens is classification into endotoxins and non-endotoxin pyrogens (Table 1). 2-4 Non-endotoxin pyrogens consist of components from Gram-positive bacteria, yeasts, molds and viruses all the way to environmental particles like rubber, plastic, dust and packaging materials. 4,5 Endotoxins include different components like lipopolysaccharide originating from Gram-negative bacteria (Fig. 2). You can read more about endotoxins in the BMG LABTECH blog What are Endotoxins?
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 |
Pyrogens, toll-like receptors 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 (Fig. 3).
Toll-like receptors are found on monocytes. They 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) from microbes including viruses and fungi and damage-associated molecular patterns arising from injured cells (Fig. 3). They also recognize danger-associated 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 transcription factors in the nucleus that lead to the production of cytokines like TNFα and the different interleukins. The cytokines that ultimately arise from the activation of toll-like receptors have a direct effect on temperature regulation in the hypothalamus. These sets of interactions allow pyrogens to trigger fever in humans and other animals.
Toll-like receptors and their specific pyrogens
As mentioned earlier, toll-like receptors are a diverse group of proteins that are specific for a wide range of ligands (Fig.3). 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 toll-like receptors on the cell surface. 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 specific routes to triggering the human fever reaction in the hypothalamus. Exogenous endotoxins like lipid A can bind to toll-like receptor 4 on the cell surface to trigger a signaling cascade that promotes endogenous cytokine release within the cell (IL-1, IL-6 or TNFα). Non-endotoxin 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.
First steps towards pyrogen testing
What is past is often a useful prologue to understand the present. 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 materials into rabbits which is the foundation of today’s rabbit pyrogen test. The large number of pyrogen tests on rabbits has driven the need for alternative tests. In the 1970s, the Limulus Amoebocyte Lysate (LAL) test, a bacterial endotoxin test, was introduced and became a widely used replacement for the rabbit pyrogen test. Further tests have been introduced including the monocyte activation test in 1995 and the recombinant factor C test in 2001. We will take a closer look at these pyrogen tests and ways to analyze endotoxins in the following section.
Methods for pyrogen tests
Four main methods are currently available for pyrogen tests and endotoxin detection.7 They are distinguished by whether they use animals for testing and their target. As shown in Fig. 4, the target can be either (a) exclusively endotoxins or (b) pyrogens more widely (defined as endotoxins and non-endotoxin pyrogens).
Rabbit pyrogen test
The rabbit pyrogen test is a broad pyrogen testing method that was the first to be developed. The test relies on the rectal measurement of body temperature after injection of the product. The rabbit pyrogen test is designed to limit the risk of a fever reaction to an acceptable level in the patient after injection of the product and can detect both endotoxin and non-endotoxin pyrogens. While the rabbit pyrogen test has been widely used over the years, it is not a quantitative assay and has low sensitivity. A significant drawback is the need for large numbers of animals to perform pyrogen tests and the inevitable suffering that accompanies the rabbit pyrogen test.
Bacterial endotoxin test or Limulus Amebocyte Lysate assay
In 1956, Fred Band discovered that the blood from the horseshoe crab coagulates to form a gel when Gram-negative bacteria or their lysates are present. This observation formed the basis for the development of the Limulus Amebocyte Lysate (LAL) assay which readily identifies the presence of bacterial endotoxins.7 Endotoxins are the main component of the outer membrane of the cell wall of Gram-negative bacteria. Most endotoxins derive from the lipopolysaccharide that is a structural component of the cell wall of these bacteria . Specifically, it is the lipid A component of lipopolysaccharide that is the toxic component of the endotoxin.
Gel-clot test, turbidimetric assay, and chromogenic method
Different LAL or endotoxin tests are available today including a gel-clot test, turbidimetric assay, and chromogenic method. The gel-clot test is simple and reliable but does not measure endotoxin concentrations quantitatively. It just confirms if endotoxin is present or not and therefore serves as a qualitative test. In contrast, the turbidimetric assay does offer a quantitative approach and measures the cloudiness or turbidity due to endotoxin binding to the amebocyte lysate. The chromogenic method is also a quantitative test that involves the release of para-nitroaniline (yellow in color) from a chromogenic substrate when hydrolysis takes place due to the pre-clotting enzyme, offering a colorimetric readout (Fig. 5). 
Both chromogenic and turbidimetric assays readily permit measurement of the amount of endotoxin in the sample. However, they cannot be used to analyze inherently colored samples like blood or urine samples. You can read more about the different types of LAL assays in the BMG LABTECH blog The LAL assay: detecting bacterial contamination.
LAL assays were quickly adopted as testing methods due to their sensitivity and specificity. However, each LAL test is specific for endotoxins only and does not test more widely for non-endotoxin pyrogens. In practice while widely used LAL assays cannot fully replace the broad applicability of the rabbit pyrogen test. Another drawback is that the test involves drawing significant amounts of blood from many horseshoe crabs to isolate the coagulation factor. This procedure has led to decreases in the horseshoe crab population in the wild.8
Recombinant factor C (rFC) test
The recombinant factor C or rFC assay is an in vitro assay that does not depend on animal use and which offers many of the benefits of the LAL assay.7 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 either a fluorescent or colorimetric product that is easily quantified (Fig. 6).
Monocyte-Activation Test
The Monocyte Activation Test (MAT) is also an in vitro assay that does not require animal use.9,10 In this case, monocytes – usually monocyte cell lines - activated by pyrogens produce cytokines (e.g. TNFα and interleukins) that can be detected in immunological assays. Different immunological assays are available including several types of enzyme-linked immunosorbent assays (ELISAs) or Lumit® Immunoassays which are a luminescence-based alternative to conventional immunoassays. Lumit® Immunoassays offer higher sensitivity and a broad dynamic range based on a simple add-mix-read protocol.
The LumiMAT™ assay is an advancement of the MAT assay and comprises a monocytic cell line engineered to include a luciferase reporter gene (Fig. 7). When these cells encounter pyrogens, the NF-κB pathway is activated leading to the expression of luciferase. Upon adding a specific substrate, the expressed luciferase catalyzes a reaction that emits luminescence. The intensity of this luminescent signal correlates with the pyrogen concentration in the sample and is measured using a luminescence microplate reader. This method offers a streamlined, single-plate assay that minimizes inter-test variability and substantially reduces handling steps.
Pyrogen tests on a microplate reader
Absorbance, fluorescence intensity, and luminescence measurements are all options for pyrogen tests. Three of the four types of tests available for pyrogen testing and described in this blog are amenable to analysis using a microplate reader. LAL or endotoxin tests, rFC assays and monocyte activation assays all benefit from the use of the cost-effective, higher throughput analysis that microplate readers can deliver. Here we consider some examples of the different detection methods for pyrogen assays on microplates and highlight distinctive features of BMG LABTECH solutions.
LAL assays
In the application note Endotoxin detection on a microplate reader using a colorimetric, kinetic endotoxin detection kit with integrated data analysis absorbance measurements were used to detect the release of p-nitroaniline (pNA) from a colorless peptide Ac-Ile-Glu-Ala-Arg-pNA. This is an example of a LAL assay with the specificity and sensitivity needed to detect low levels of bacterial endotoxin. In the presence of endotoxin, a proenzyme in the LAL is activated which cleaves the colorless peptide to yield a colored product that can be measured over time (Fig. 8). In the study, a Lonza endotoxin detection kit was used on a BMG LABTECH microplate reader and data were analyzed using the MARS data analysis software. The time needed to reach a defined absorbance is then plotted against the known endotoxin concentrations. Predefined functions in MARS provide effortless data analysis including the calculation of the reaction times, plotting of standards and calculation of concentrations of unknown samples. The Kinetic-QCL™ Assay for endotoxin detection is compatible with all BMG LABTECH microplate readers including the SPECTROstar® Nano, PHERAstar® FSX, the Omega series, VANTAstar® and CLARIOstar® Plus.
The presence of an ultrafast UV/vis spectrometer is a useful asset for absorbance-based measurements required for LAL assays. The kinetic detection mode of BMG LABTECH’s readers enables time-resolved observation of the signal increase in real-time as an assay goes to completion without further intervention. Due to the ultra-fast speed of spectral capture, time-course experiments for changes in absorbance can be readily monitored with high data density. Absorbance detection with the ultra-fast UV/vis spectrometer is available on all BMG LABTECH’s single- and multi-mode microplate readers except for the NEPHELOstar Plus.
rFC assays
rFC assays are available with colorimetric or fluorogenic readout. In the application note Faster PyroGene™ Detection of Endotoxin using Enhanced Dynamic Range on the CLARIOstar Plus a fluorescent rFC assay was used for endotoxin testing. Fluorescent intensity detection using the PyroGene™ Recombinant Factor C assay from Lonza allows for rapid kinetic detection of endotoxins and provides a good alternative to LAL-based detection. As mentioned earlier, LAL tests have repercussions for the sustainability of horseshoe crab populations in the wild. This application note demonstrates the value of the Enhanced Dynamic Range (EDR) feature on the CLARIOstar Plus which ensures accurate signal quantification across low to high concentrations of analyte, in this case endotoxin without running into the risk of signal saturation. Both the CLARIOstar Plus and the VANTAstar as well as the PHERAstar FSX include Enhanced Dynamic Range technology for superior performance in a single luminescence or fluorescence run.
The CLARIOstar Plus and VANTAstar additionally offer outstanding wavelength flexibility, which is an asset for rFC and other assays . Increased light transmission and sensitivity is possible courtesy of Linear Variable Filter MonochromatorsTM and different filter options.
Additional microplate reader features like incubation, shaking and an Atmospheric Control Unit provide distinct benefits for pyrogen assays. The enzymatic cascades that are the central element of pyrogen testing assays like LAL or rFC have a temperature optimum at 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 (some optionally up to 65°C).
The available shaking options support users in the proper mixing of assay reagent before the kinetic monitoring starts.
The Atmospheric Control Unit from BMG LABTECH provides researchers with a system that enables control of both the oxygen and carbon dioxide concentrations in an independent manner. Consistent stirring options also deliver benefits for assays where this is critical. This accessory is especially relevant when it comes to application of cell-based assays as it transforms the reader into an incubator. The ACU allows to monitor the development of the luminescent signal derived from the reporter cell line in LumiMAT assays over time in the microplate reader, instead of only observing the endpoint signal after the predefined incubation time. The VANTAstar®, the CLARIOstar® Plus, and the Omega series can be combined with the Atmospheric Control Unit.
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 existing 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.
Industry-wide applications for pyrogen tests
New innovations for pyrogen tests that deliver safety and scale will bring further savings in costs and time and will benefit from the automation friendly approaches offered by microplate readers.
As highlighted in this blog, instead of elaborate multi-step processes like incubating cells with samples and using the supernatant for immunoassays, the LumiMAT™ assay delivers an add-mix-read approach that significantly reduces handling steps and simplifies the transfer to automated systems. The availability of options like the ENDOZYME® II GO kit, a ready-to-go microplate precoated with standard and positive controls, 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 in turn ensure further innovation and provide incentives for improved tests for pyrogens. As such, pyrogen tests will be relevant to many products arising from biotechnology, drug discovery, microbiology, immunology and many other areas of quality control and bioanalysis.
BMG LABTECH solutions
What is the preferred BMG LABTECH microplate reader for specific needs and applications related to the determination of pyrogens? Absorbance detection is available on BMG LABTECH’s complete portfolio of microplate readers with the ultra-fast spectrometer.
BMG LABTECH also offers a range of multi-mode detection devices for sensitive fluorescence and luminescence measurements.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus, and PHERAstar® FSX microplate readers optionally 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.
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
- Pyrogens, Still a Danger | FDA https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/pyrogens-still-danger. Accessed 03/20/2025
- 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.
- 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.
- 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. Epub 2020 May 16.
- 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).
- Hort, E,. Penfold, W.J., A Critical Study of Experimental Fever. Lister Institute of Preventive Medicine, March 14, 1912.
- 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.
- 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.
- 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.
- 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.
