Introduction
Pyrogen contamination in pharmaceuticals, biologicals and medical devices poses a serious risk to health that requires reliable detection methods to ensure patient safety. Tradition-al tests, such as the Rabbit Pyrogen Test and Limulus Ame-bocyte Lysate (LAL) assay, come with ethical concerns due to animal use, limited sensitivity, and labour-intensive protocols 1. The Monocyte Activation Test (MAT) typically uses a monocytic cell line which is activated by pyrogens and produces inflammatory cytokines upon activation. The incubation time of the cells with the samples and the subsequent quantification of released cytokines with immunological assays like ELISAs (Enzyme-Linked Immunosorbent Assays) typically takes ~ 1.5 days 2. The LumiMAT™ assay delivers detection of endotoxin and non-endotoxin pyrogens by leveraging a luciferase-based MAT, providing rapid, highly sensitive, and animal-free results in just 5 hours 3. When performed on BMG LABTECH microplate readers, LumiMAT benefits from high-throughput capabilities, effortless automation, exceptional reproducibility, and streamlined workflows. This application note highlights the powerful synergy between LumiMAT and detection on BMG LABTECH readers.
Assay principle
The LumiMAT assay uses a monocytic cell line (NOMO-1) that has been genetically modified to include a luciferase reporter gene (Fig. 1). The luciferase protein is produced in response to NF-κB signals activated by exposure to pyrogens.
Materials & methods
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LumiMATTM Pyrogen Detection Kit, Reagent Set + Cells (#297-96801 & 298-36991, Fujifilm Wako)
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Control Standard Endotoxin (CSE, #CSE4037-5006, Fujifilm Wako) or Standard Endotoxin (JP-RSE, #1032001021, Pharmaceutical and Medical Device Regulatory Science Society of Japan)
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96-well plate, white, tissue-culture (TC)-treated (#655073, Greiner BioOne) or 96-well fl at bottom plate TC-treated (#136101, Nunc)
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BET-qualified lab materials (pipette tips, sample and reagent tubes)
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Automated dispenser pipette
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VANTAstar® with Atmospheric Control Unit (ACU) and FLUOstar® Omega
Experimental Procedure
Standards and samples were diluted with the LumiMAT dilution medium according to the manufacturer’s instruction manual. 50 µL of standards, samples, the blank (dilution medium only), and the negative control (endotoxin-free water) were transferred to the wells of a white TC-treated 96-well microplate in quadruplicate. The reporter cell line NOMO-1 was thawed, diluted in 9.5 mL LumiMAT assay medium and centrifuged at 300 g for 5 min. The cell pellet was resuspended in 5.5 mL of assay medium and 50 µL of cell suspension was transferred to all standard, sample and blank wells. The lidded plate was transferred to the VANTAstar, which was set to 37 °C and 5 % CO2 30 min earlier.
A protocol with a preset for automatic removal of the microplate after 3 h ensured that the incubation time was precisely met (only available on the VANTAstar and the CLARIOstar® Plus). 12 mL luciferase buffer was combined with 240 µL luciferase substrate. After the priming of the injector with the luciferase-substrate-buffer mix, the plate lid was removed, 100 µL of the mix were injected in each well and the wells were evaluated with the following settings.
Instrument Settings
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Luminescence, plate mode
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Optic settings |
Filters |
Full signal |
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General settings |
Settling time |
0.02 s |
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Integration time |
0.8 s |
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Gain settings |
EDR* or 3600 |
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Kinetic settings |
Number of cycles |
2 |
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Cycle time |
3 min |
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Injection settings |
Injection volume |
100 μL/well |
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Pump speed |
225 μL/s |
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Incubation |
37 °C, 5 % CO2 |
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Results & Discussion
The measured luminescence values (relative luminescence units RLUs) of a standard series of CSE prepared on the TC-treated 96-well plate from Greiner were used to create a 4-parameter fit using using BMG LABTECH’s MARS Data Analysis Software (Fig. 2).
The 4-parameter fit (Fig. 2) resulted in a correlation coefficient of 0.9995, which exceeds the quality criteria specified by the manufacturer of the LumiMAT kit (R² > 0.975). The R² values to-gether with the low standard deviations (SD) of the individual standards indicate robust and reliable measurements. The Signal to Blank ratios (S/B) of S1 and S7 were 98.2 and 3.9, respectively, which confirms a very good data quality. The evaluation of the LumiMAT kit with the FLUOstar Omega provided the following values: R² = 0.9999, S1/B = 44.2, S7/B = 1.8). Collectively, these values emphasise the excellent dynamic range that can be achieved using the kits and BMG LABTECH devices.
The curve for the 4-parameter fit was then used to calculate the pyrogen concentration of the unknown samples (Fig. 3). 
The addition of known amounts of pyrogen (pyrogen spikes) to unknown sample wells allows for determination of the spike recovery. The spike recovery can be used to check whether sample components interfere with the LumiMAT assay. Possible sample interference can then be indicated by a failed recovery rate. These calculations and validation of the sample spike recovery rates are easily performed using MARS (Table 1). In this example, glucose injection samples were tested for endo-toxins using the LumiMAT kit together with the JP-RSE and the TC-treated 96-well plate from Nunc on the FLUOstar Omega.
Table 1: Recovery rate of pyrogen spikes measured with the FLUOstar Omega.
| Content | Recovery Rate Spikes (%) | Recovery Rate (%) |
| Sample 7 | 123.1 | passed |
| 126.9 | passed | |
| 102.5 | passed | |
| 129.2 | passed | |
| Sample 8 | 88.4 | passed |
| 80.5 | passed | |
| 72.7 | passed | |
| 80.7 | passed |
Conclusion
The LumiMAT assay is ideally suited for the measurement of endotoxin and non-endotoxin pyrogens and improves their detection compared with conventional MAT assays and other pyrogen assays. A gene reporter assay allows luciferase to be expressed in response to NF-kB signals activated by exposure to pyrogens. This luminescence-based gene reporter assay significantly accelerates the evaluation procedure compared with the traditional MAT test and offers higher sensitivity. Its fast and highly sensitive performance combined with BMG LABTECH reader’s precise, streamlined operation, delivers robust and reliable results, simplifying workflows and improv-ing overall efficiency. In addition, the flexibility of adding an ACU and injectors to BMG LABTECH’s microplate readers offer further options to adapt the workflow as needed and can be an alternative to purchasing an incubator.
References
- Maloney et al. (2018) Saving the horseshoe crab: A synthetic alternative to horseshoe crab blood for endotoxin detection. PLoS Biol. 16(10):e2006607. doi: 10.1371/journal.pbio.2006607.
- Hartung T. (2021) Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test. ALTEX. 38(1):3-19. doi: 10.14573/altex.2101051.
- Rapid Microbiology (2024) LumiMAT™: Rapid and reliable pyrogen detection assay for MAT testing. Rapid Microbiology.