Introduction
Reliable endotoxin testing is essential to ensure the safety of pharmaceuticals, biologics and medical devices. Different methods are available for detecting endotoxins, including the rabbit pyrogen test, the Limulus Amoebocyte Lysate (LAL) assay or monocyte activation tests with subsequent cytokine detection. Alongside the fact that animal-based methods present challenges in terms of reproducibility, specificity, and operational workload, there is growing ethical concern and increasing pressure to minimize animal use and replace it with synthetic alternatives wherever possible. The approach based on recombinant Horseshoe Crab Factor C (rFC) eliminates the need to harvest the animal’s blood, from which the lysate is traditionally obtained for endotoxin detection in conventional LAL assays. In addition, rFC-based assays offer improvements in specificity, lot-to-lot consistency and savings in costs and resources while providing a sustainable, animal-saving, and secure alternative compared to conventional assays1.
The ENDOZYME® II assays from bioMérieux are based on rFC and provide a fluorescence readout. The underlying methodology of the kits is accepted by the FDA2, included in the European Pharmacopoeia (chapter 2.6.32) since 20213 and adopted by the US Pharmacopeia as of May 2025 in the new chapter 864.
ENDOZYME II is either available for manual microplate preparation or as the ENDOZYME® II GO kits with the control standard endotoxin (CSE) already pre-coated on either plates or strips for a maximum efficiency and flexibility. All ENDOZYME II kit configurations are based on the same assay principle.
BMG LABTECH readers with Enhanced Dynamic Range (EDR) offer significant advantages to simplify the measurement workflow. EDR eliminates the need for a preliminary run to determine the gain, as it automatically measures each well with the optimal gain. In addition, both very high and very low signals can be readily detected on the same plate in the same run. This application note describes endotoxin testing with ENDOZYME II on the VANTAstar® as part of a validation process at CODAN Medizinische Geräte GmbH.
Assay principle
ENDOZYME II is a homogeneous enzymatic assay that uses synthetic rFC in combination with a fluorogenic substrate.
The binding of endotoxins by rFC results in an active form of the enzyme that cleaves the fluorogenic substrate to release the detectable fluorophore. The fluorescence increase over time is proportional to the endotoxin concentration.
Materials & methods
- BET-qualified lab materials (pipette tips, sample and reagent tubes)
- Automated dispenser pipettes
- ENDOZYME II (bioMérieux)
- VANTAstar (BMG LABTECH)
Experimental Procedure
Plate preparation
For the manual microplate preparation, CSE was reconstituted and diluted according to the instructions for the ENDOZYME II kit to create a standard series. Samples, standards and blanks (endotoxin-free water) were transferred to the plate at 100 µL/well. 0.5 EU/mL CSE Positive Product Controls (PPC) were included in duplicate for each sample to check if sample components interfere with the assay.
The ENDOZYME II GO version of the kit alternatively provides users with the ready-to-use GOPLATE™ system. GOPLATEs are 96-well plates precoated with precise amounts of CSE for the standard curve and PPCs. No manual dilution series and no loading of the PPCs is necessary. Precoated wells are simply reconstituted by adding 100 µL endotoxin-free water. The use of GOPLATEs reduces the necessary handling steps, decreases accompanying data variability, and avoids invalid outcomes like false positive results. The following steps are independent of the kit version. The ENDOZYME II microplate including standard and samples was subsequently transferred to the preheated microplate reader at 37 °C and left to equilibrate for 5 min. In the meantime, the assay reagent was prepared by combining eight parts of assay buffer with one part of enzyme and one part of substrate. 100 µL assay reagent was added to all wells to be analysed. The microplate was shaken (double-orbital mode) in the reader for 15 s at 300 rpm and the measurement for t0 was started immediately afterwards using the settings shown in the table below. The second measurement was performed after 20 or 60 min depending on the desired sensitivity (0.05 or 0.005 EU/mL).
Instrument settings (for 60 min protocol, 0.005 EU/mL)
|
Fluorescence Intensity, plate mode
|
||
|
Optic settings
|
LVF |
380-15
445-20 |
|
General settings
|
Number of Flashes |
40
|
| Settling time |
EDR |
|
|
Gain
|
4.8 |
|
|
Shake
|
Shake before first cycle | |
|
Shaking pattern
|
Double orbital |
|
|
Shaking frequency
|
300 rpm |
|
|
Time
|
15 s |
|
|
Kinetic settings
|
Number of cycles
|
2 |
|
Cycle time
|
60 min |
|
|
Incubation
|
37 °C |
|
Results & Discussion
Fluorescence readings (expressed as relative fluorescence units: RFUs) for the t0 measurement of the ENDOZYME II assay were subtracted from the fluorescence of the t60 measurement and afterwards corrected for the blank. The subsequent difference in fluorescence values (dRFUs) was fitted against the concentrations of the standard series (Fig. 2) and resulted in a linear correlation with an R² of 0.99997.
The regression fit was used to calculate the endotoxin concentrations of the unknown samples (Fig. 3) and the recovery of PPC in spiked samples (Fig. 4). 
Calculations and validations of the ENDOZYME II assay results were performed using BMG LABTECH’s MARS analysis software. The data visualisation enables quick identification of samples that do not meet the validation criteria (Fig. 4). The creation of templates allows all calculation steps to be easily transferred to upcoming measurements.
Fig. 4: Validation of PPC recovery in the range of 50% to 200% with MARS data analysis software.
| Content | Group | PPC recovery [%] | PPC recovery |
| Sample X1 | A | 104.5 | valid |
| Positive control P | |||
| Sample X2 | B | 87.3 | valid |
| Positive control P | |||
| Sample X3 | C | 92.2 | valid |
| Positive control P | |||
| Sample X4 | D | 92.1 | valid |
| Positive control P | |||
| Sample X5 | E | 106.6 | valid |
| Positive control P | |||
| Sample X6 | F | 95.4 | valid |
| Positive control P | |||
| Sample X7 | G | 100.4 | valid |
| Positive control P |
Conclusion
Advances in endotoxin detection using rFC-based assays have set a new benchmark in precision and reliability. Compared to conventional methods, these assays offer unparalleled specificity, flexibility, and lot-to-lot consistency, and deliver sustainability through their animal-saving technology. The use of rFC assays together with BMG LABTECH’s EDR technology, streamlines and accelerates the workflow and delivers single step evaluation. The combination of ENDOZYME II kits and detection on the VANTAstar ensures rapid and accurate results. This synergy saves valuable time and paves the way for efficient high-throughput testing. By using the ENDOZYME II GO system with pre-coated CSE plates, the time required for analysis can be further reduced. Together, these advantages simplify endotoxin testing, offering a robust, sustainable, and reliable solution for modern laboratories.
References
- Gauvry, G.A., Uhlig, T., Heed, K. (2022). Efforts: 2007-2020. Springer, Cham. https://doi.org/10.1007/978-3-030-82315-3_21
- US FDA (2012) Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers.
- European Pharmacopoeia Supplement 10.3 (2020), general chapter 2.6.32. Test for bacterial endotoxins using recombinant factor C
- Expert Committee approves endotoxin testing using non-animal derived reagents (2024), Press release: https://www.usp.org/news/expert-committee-approves-endotoxin-testing-using-non-animal-derived-reagents