Endotoxin (LAL) reader
An incubating photometer that holds LAL reactions at 37 °C and measures the change in absorbance or turbidity over time. Software turns the reaction onset time into an endotoxin concentration in EU/mL using a standard curve.
- Pharmaceutical
- Biotech
- Medical devices
- Dialysis and hospital pharmacy
- Research laboratories
What is Endotoxin reader?
Bacterial endotoxins are lipopolysaccharides from the outer membrane of Gram-negative bacteria. They cause fever and shock if injected, so parenteral products and devices are tested for them. The test uses Limulus amebocyte lysate (LAL), a reagent from horseshoe crab blood that reacts with endotoxin in an enzyme cascade.
An endotoxin reader incubates LAL reactions at 37 ± 1 °C and reads them continuously. In the kinetic chromogenic method the cascade releases a yellow dye (p-nitroaniline) read at 405 nm. In the kinetic turbidimetric method the reaction forms a gel and the turbidity increases. The time taken to reach a set absorbance (the onset time) is shorter when there is more endotoxin.
Readers are either 96-well incubating microplate readers or multi-channel tube readers. Software builds a log-log standard curve of onset time against endotoxin concentration and calculates the endotoxin content of samples and the recovery of spiked samples. The gel-clot method does not need a reader, only a 37 °C heating block or water bath.
Used for
- Bacterial endotoxins test (BET) of parenteral products, WFI and APIs
- Testing of medical devices and container closures (rinse extracts)
- Depyrogenation validation: endotoxin indicator recovery after dry heat
- In-process monitoring of water systems and bioprocesses
- Inhibition / enhancement testing during method validation
How it works
- 01
Prepare
Standards, samples, spiked samples and negative controls are made in LAL reagent water using pyrogen-free tips and tubes.
- 02
Pre-warm
The plate or tubes are pre-incubated at 37 °C in the reader.
- 03
Add LAL
Reconstituted LAL reagent is added to every well, quickly and in a fixed order.
- 04
Read kinetically
Absorbance (405 nm) or turbidity is read at short intervals while held at 37 °C.
- 05
Onset time
Software records the time each well reaches the set optical density.
- 06
Calculate
Onset times are converted to EU/mL via the log-log standard curve; recovery of spikes is calculated.
Advantages
- Quantitative result over a wide range (often 0.005–50 EU/mL)
- Low limit of detection compared with gel-clot
- Automated reading and calculation with audit trail
- Many samples and replicates in one run (microplate)
Limitations
- Results depend on accurate temperature control and pipetting
- Products may inhibit or enhance the reaction and need dilution and validation
- Glucans can give false positives with some LAL reagents
- Software and reader need computer system validation
- Reagent lots and endotoxin standards must be matched and qualified
1 calibration procedure
Methods that use Endotoxin reader
Keep every Endotoxin reader in control: usage logs, calibration and maintenance schedules, and automatic blocking of overdue instruments.