PHERAstar FSX
Powerful and most sensitive HTS plate reader
Introduction
The recombinant factor C (rFC) test is an in vitro method used to test for bacterial endotoxins (lipopolysaccharides), an often-encountered type of pyrogen.1,2 rFC is a genetically engineered protein that is activated by endotoxin and is usually based on a fluorescent readout. Once activated, the rFC enzyme releases a fluorescently labeled product from an engineered substrate that is quantifiable and proportional to the amount of endotoxin present.
rFC testing is designed to ensure the safety of pharmaceuticals, biologics and medical devices that are introduced into humans or other animals. It serves as an alternative to the Limulus Amebocyte Lysate (LAL) assay and plays a crucial role in many quality control and bioanalysis processes.3
Accurate measurement of endotoxins is crucial to ensure reliability and compliance in many processes. For example, microorganisms can contribute to endotoxin contamination during sterilization processes, as endotoxins are heat-stable components of the cell walls which do not only survive the sterilization process, but are even increasingly released. Inactivation of endotoxins during manufacturing, sterilization, and disinfection processes remains a significant challenge. Emerging technologies, such as cold plasma treatment, enzymatic degradation or nanomaterial-based approaches, show promise for endotoxin inactivation, but even 'sterile' devices can harbor endotoxins, posing safety risks. Preventing endotoxin contamination is crucial to ensure safety in medical environments. Controlling endotoxin levels in solutions used for instrument cleaning and rinsing is an essential part of this process.
The rFC test provides a more sustainable alternative to traditional animal tests or animal-derived test components. The adoption of rFC in several industries highlights its benefits in ensuring patient safety and its alignment with animal conservation efforts.
In this blog, we look at the rFC test and examine how microplate readers can help support this test for endotoxins.
Endotoxins and their effects on the immune system
Endotoxins are a type of pyrogen, a diverse group of substances that can result in adverse effects in the body due to increases in body temperature. Pyrogens can range from chemical substances (e.g. plastic, rubber or packaging materials, examples of non-endotoxin pyrogens) to substances from Gram-positive bacteria (e.g. lipoteichoic acid, peptidoglycans, further examples of non-endotoxin pyrogens) or substances from Gram-negative bacteria (lipopolysaccharides). Endotoxins are the lipopolysaccharide component of the outer membrane of the cell wall of Gram-negative bacteria. They are widely distributed in the environment and are amongst the most potent pyrogens encountered. The core polysaccharide, a structural component of lipopolysaccharides, plays a significant role in immune recognition and variability among different bacterial species and strains. In addition, the length of the O-antigen component of endotoxins can vary significantly among different species and strains of Gram-negative bacteria, influencing its immunogenic properties and recognition by antibodies.
The presence of endotoxins in various environments is significant as they are recognized by the immune system as a threat, leading to various physiological responses and complications during infections. Endotoxins can enter the blood stream, triggering severe immune responses. This immune response can escalate to sepsis, a severe condition that can result in life-threatening complications.
If you are interested in more details about the characteristics and effects of endotoxins, please take a look at our blog article “What are endotoxins”.
Triggers of fever
Inflammation is a critical response to endotoxins from Gram-negative bacteria. Neutrophils play a crucial role in mediating the immune response to endotoxins, contributing to inflammation and the overall defense against bacterial threats. These endotoxins can provoke an immune response leading to inflammation and ultimately fever, as the innate immune system recognizes them as threats, resulting in the secretion of pro-inflammatory cytokines and other immune reactions.
What is rFC testing?
Over time, different tests for pyrogens have been developed (Fig. 1) each with unique capabilities and features. Broadly speaking, pyrogen tests can be divided into two groups: those that permit testing for a complete range of pyrogens (i.e. bacterial endotoxins and non-endotoxin pyrogens) and those that test for bacterial endotoxins only, like the LAL assay.
Two methods are commonly used for endotoxin detection based on LAL: a clotting reaction involving a coagulable protein found in Limulus polyphemus and a more sensitive photometric test.
Compared to other available tests (Fig. 1), the rFC test functions as an alternative to those methods that involve animal testing. It also offers high sensitivity and is cost effective. Like LAL assays, the rFC test is specific for endotoxins and does not detect non-endotoxin pyrogens (Table 1). Endotoxin detection can occur in various scenarios, emphasizing the importance of using a reliable test like rFC.
Table 1. Features of rFC test versus other pyrogen/endotoxin tests.
| rFC test | Rabbit pyrogen test |
LAL test | Monocyte Activation Test | |
| Type of pyrogen tested | Endotoxins (lipopolysaccharides) only | All pyrogens (endogenous and exogenous) | Endotoxins (lipopolysaccharides) only | All pyrogens (endogenous and exogenous) |
| Biological material or animal used | rFC | Live rabbits | Amebocyte lysate from horseshoe crabs | Human blood-derived monocytes, monocytic cell lines |
| Ethical concerns for animal use for test | No | Yes | Yes | No |
| Animal source of test components | No | Not applicable (animal is the test) | Yes | Yes (human blood-derived components) |
| Microplate assay | Yes | No | Yes | Yes |
| Detection mode | Fluorescence (absorbance also an option for rCR) | Not applicable | Absorbance, turbidimetric | Absorbance, fluorescence, luminescence |
| Sensitivity | High for endotoxins | Moderate for all pyrogens | High for endotoxins | High for all pyrogens |
| Specificity | Specific to endotoxins | Non-specific | Specific to endotoxins | Broad specificity |
| Regulatory status | Accepted by European and US Pharmacopeia | First test to be adopted and accepted for use by European and US Pharmacopeia | Widely accepted (European, US, and Japanese Pharmacopeia | Recognized by European Pharmacopeia for advanced applications |
Specifics of the rFC test
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 in most cases from a substrate that is easily quantified. 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 rFC test's counterpart: The LAL assay
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. It is essential to validate the rFC test to ensure its effectiveness in detecting endotoxins.
Both the LAL and rFC tests rely on factor C as part in the detection process. However, unlike rFC, 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. The rFC test only has the factor C pathway. It therefore cannot be activated by β-glucans (Fig. 2) which gives it an advantage as an endotoxin test.
Lipids, particularly lipid A, play a crucial role in the structure of endotoxins found in Gram-negative bacteria. Lipid A, a hydrophobic component of lipopolysaccharide (LPS), contributes to the structural integrity and protective functions of the bacterial outer membrane, enabling resistance to antimicrobial compounds and immune responses. You can read more about lipid A in the BMG LABTECH blog What are Endotoxins?
For many years, LAL has been the method traditionally used for endotoxin testing. However, it relies on the use of blood samples taken from the horseshoe crab. Two main species have been used as sources: Limulus polyphemus from the North Atlantic and Tachypleus spp. from Asia. Blood is collected in both cases from live animals and used as a source of an amebocyte-rich fraction (amebocytes are cells found in invertebrates that play a role in the defense against pathogens). The amebocytes are lysed to release the coagulation proteins that are vital for the LAL test.
Unwanted 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 since a significant number of animals die either during blood collection or when they are released back into the wild. The deaths of horseshoe crabs are estimated to be around 15% when they are released back to the wild after approximately 20% of blood is withdrawn as a source of factor C. 4 Methods like the rFC test that do not depend on animal sources are therefore attractive alternatives to the LAL 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.
rFC tests on a microplate reader
rFC tests benefit from the use of the cost-effective, higher throughput analysis that microplate readers can deliver. For rFC tests, fluorescence intensity measurements are the method of choice to determine endotoxin levels accurately.
Here we consider some examples of applications for rFC tests on microplates and highlight distinctive features of BMG LABTECH solutions.
The PyroGene™ Recombinant Factor C assay
The application note Faster PyroGene™ Detection of Endotoxin using Enhanced Dynamic Range on the CLARIOstar Plus describes a fluorescent rFC assay amenable to analysis on a microplate reader (Fig. 3).
As we saw earlier, factor C is a protease whose normal function is to serve as an initiator of the limulus coagulation cascade after exposure to endotoxin (lipopolysaccharide). Its regular substrate is factor B and knowledge of the cleavage site in factor B has enabled the production of artificial substrates that can be detected in in vitro assays. In the PyroGene™ Recombinant Factor C assay from Lonza, binding of rFC to endotoxin results in an active form of the enzyme. This activated form of the rFC enzyme subsequently cleaves a fluorogenic substrate which releases the detectable fluorophore. Fluorescent intensity detection using PyroGene™ allows for rapid kinetic detection of endotoxins and provides a good alternative to LAL-based detection.
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. Low and high concentrations of endotoxin can be readily measured without running into the risk of signal saturation. The CLARIOstar Plus, VANTAstar® and PHERAstar® FSX all include Enhanced Dynamic Range technology for superior performance in a single fluorescence run.
The ENDOZYME® II rFC assay
In the application note Streamlined detection of endotoxin using the ENDOZYME® II recombinant factor C assay and Enhanced Dynamic Range on the VANTAstar®, a rFC test from BioMérieux was used to decrease handling time and improve workflow efficiencies for faster and more consistent endotoxin testing. The assay was performed on the VANTAstar and, like in the previous example, made use of the Enhanced Dynamic Range (EDR) feature which helped to streamline and accelerate the workflow. The signal intensities of samples in enzymatic kinetic assays are often hard to predict as the maximum signal builds up over time during the kinetic assay. This is also the case in the ENDOZYME II assay, which typically requires a preliminary run to determine the optimum gain for subsequent measurements. EDR enables the optimum gain to be determined in the same test run, thus eliminating the need for additional runs saving both time and material.
In this example, the ENDOZYME® II recombinant factor C assay (ENDONEXT™ technology) was performed on two different plates. For conventional plates, the standard is prepared and titrated externally. The GOPLATE™ includes wells already precoated with control standard endotoxin (CSE) for the standard curve and positive control wells supporting the evaluation of spike recovery in unknown samples. The precoated plates thus reduce the amount of manual work needed and offer maximum efficiency. Calculations and validations of the assays were processed using BMG LABTECH’s MARS analysis software. Data visualization enabled rapid identification of samples that did not meet validation criteria. The ability to create assay templates permitted calculation steps to be readily transferred to further measurements.
In all cases, the fluorescent ENDOZYME® II rFC assay offers an environmentally sustainable method for endotoxin testing geared to pharmaceutical, biologics and medical device quality control. Clear benefits are 100% endotoxin specificity, lot-to-lot consistency, and more streamlined workflows. The ENDONEXT™ technology used in the ENDOZYME® II rFC assay provides reliable results for many aspects of endotoxin measurement ranging from in-process controls to final product testing on the most complex matrices. All assays are validated according to European and US Pharmacopeia.
Advances in rFC testing
Traditional endotoxin testing methods often require heavy manual preparation leading to inefficiencies, increased costs, and inaccurate results. The rFC tests described in this blog, when combined with the capabilities of a microplate reader, 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 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) 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 yellow-colored product (para-nitroaniline) when cleaved by the final activated enzyme in the rFC cascade. Detection is by absorbance measurements.
Further trends in rFC tests
Several factors will continue to shape trends in endotoxin testing and, more widely, the testing for all types of pyrogens. The move away from animal-based tests is much needed and is driven by ethical concerns and changes in the regulatory environment. It will continue to gather pace. This makes methods like rFC tests an excellent starting point for further innovation.
Other areas for refinement include performance and applicability. For example, some of the currently available rFC tests may be prone to interference due to environmental conditions. Further innovation in rFC technologies may help to produce optimized proteins that are more robust to changes in pH and ionic strength, for example, or which offer improved recovery of endotoxins or less masking of signals of endotoxins by other constituents.
rFC tests, like LAL tests, are restricted in the range of source material that they can be applied to due to possible assay interference from different substances in the starting material. For example, they cannot be used to detect endotoxin in serum, plasma and blood samples due to strong interference from certain blood components including proteins, lipids, cells and ions. Interference for rFC tests is also possible from detergents or salts in buffers.
Further benefits will arise if ways are found to extend the range of applicability of recombinant proteins for endotoxin testing. The EndoLISA® test 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.
As innovation proceeds, rFC tests will become increasingly relevant to more products arising from biotechnology, drug discovery, microbiology, immunology and many other areas of quality control and bioanalysis. Effective solutions for endotoxin detection will be crucial in ensuring the quality and safety of these products.
Note: The rFC test was accepted in the US Pharmacopeia and was officially recognized as such in May 2025. It has been available in the European Pharmacopeia since 2020 and permitted for use since its inclusion.
BMG LABTECH solutions
What is the preferred BMG LABTECH microplate reader for specific needs and applications related to the determination of rFC? BMG LABTECH offers a range of fluorescence plate readers for sensitive measurements of rFC assays. These include the Omega series, CLARIOstar Plus, VANTAstar and PHERAstar FSX. The capability of these readers can be further expanded with multi-mode detection including for instance absorbance and luminescence detection to also cover LAL and monocyte activation tests. The CLARIOstar Plus and VANTAstar additionally offer outstanding wavelength flexibility, which is an asset for other assays. Increased light transmission and sensitivity are possible courtesy of Linear Variable Filter MonochromatorsTM and different filter options. The EDR function available for the CLARIOstar Plus, the VANTAstar and the PHERAstar, automates gain adjustment, eliminating the need for pre-set signal amplification and reducing repeated measurements to find the optimal range.
Additional microplate reader features like incubation and shaking provide further benefits for rFC tests. The enzymatic cascades that are the central element of rFC testing 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 (optionally up to 65°C). The available shaking options support users in the proper mixing of assay reagent before the kinetic monitoring starts.
All BMG LABTECH microplate readers have exceptionally fast reading capabilities. In addition, the Omega series, CLARIOstar Plus, and PHERAstar FSX microplate readers come with on-board injectors that can offer the very best options for detection at the time of injection. The VANTAstar can be equipped with a modular injection unit.
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
- 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.
- Schneier M, Razdan S, Miller AM, Briceno ME, Barua S. Current technologies to endotoxin detection and removal for biopharmaceutical purification. Biotechnol. Bioeng. 2020 117(8):2588-2609. doi: 10.1002/bit.27362.
- 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.
- Maloney T, Phelan R, Simmons N. Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLoS Biol. 2018: 16(10):e2006607. doi: 10.1371/journal.pbio.2006607.
- Das AP, Kumar PS, Swain S. Recent advances in biosensor-based endotoxin detection. Biosens. Bioelectron. 2014 51:62-75. doi: 10.1016/j.bios.2013.07.020.
