What is endotoxin?

Endotoxin is a lipopolysaccharide (LPS) component of the outer membrane of Gram-negative bacteria and is a strong pyrogen.

• Relevant for peptides used in sensitive assays and downstream biological applications
• Measured as endotoxin units (EU) under defined test conditions
• Requires careful control of sample matrix effects to avoid invalid results

Why this matters for peptide or oligo programs

• Safety & performance: contaminants can trigger inflammatory responses or confound in vivo / cell-based readouts.
• Data integrity: glucan can produce an apparent endotoxin signal in LAL assays, risking out-of-spec outcomes.
• Decision readiness: your result must be interpretable—valid dilution, appropriate controls, and clear reporting.

Testing approaches

Qualitative endotoxin screening

A qualitative screen provides a fast “presence/absence style” assessment (as scoped) to help determine whether a sample needs further quantitative work or additional dilution/interference investigation.

• Useful for early-stage risk screening
• Guides follow-up quantitative design when needed
• Interference risk reviewed based on sample matrix

Quantitative endotoxin measurement

Quantitative testing reports endotoxin levels in EU with defined conditions. A validated dilution is selected using a dilution series so results fall within an appropriate measurement window and meet validity criteria (scope-dependent).

• EU reporting (e.g., EU/mL or EU/mg; as scoped)
• Dilution series helps identify a valid testing dilution
• Optional interference checks aligned to compendial expectations (scope-dependent)

Endotoxin Testing Methods: Gel-Clot & Kinetic Turbidimetric

We provide endotoxin testing using two established Limulus Amebocyte Lysate (LAL)-based methodologies: Gel-Clot (qualitative) and Kinetic Turbidimetric (quantitative). Method selection depends on the intended application, required sensitivity, and reporting format.

1. Gel-Clot Method (Qualitative)

The Gel-Clot method is the original compendial LAL assay and remains widely used as a qualitative limit test for endotoxin detection. It is based on the natural coagulation cascade of horseshoe crab (Limulus) amebocyte lysate in the presence of endotoxin.

Principle of the Method

When endotoxin is present at or above a defined sensitivity threshold, it activates a proteolytic cascade within the LAL reagent, resulting in the formation of a firm gel. After incubation at controlled temperature, the reaction tube is inverted:

• Firm gel remains intact → Positive result
• No gel formation → Negative result

Key Characteristics

• Qualitative “pass/fail” limit test
• Sensitivity defined by reagent label (e.g., λ value)
• Compendial recognition (USP <85>, EP 2.6.14)
• Simple and robust when matrix interference is controlled

When Gel-Clot Is Appropriate

• Limit testing against a predefined endotoxin specification
• Screening where numeric EU value is not required
• Regulated non-GMP documentation aligned to compendial methods

Gel-Clot provides clear limit-based decisions but does not generate a numeric endotoxin concentration.

2. Kinetic Turbidimetric Method (Quantitative)

The Kinetic Turbidimetric method provides quantitative endotoxin measurement by continuously monitoring the increase in turbidity that occurs during the LAL clotting reaction.

Principle of the Method

As endotoxin activates the LAL cascade, a clotting enzyme forms, increasing solution turbidity. The instrument measures optical density over time. The time required to reach a defined turbidity threshold is inversely proportional to endotoxin concentration.

A standard curve constructed from known endotoxin standards allows calculation of endotoxin levels in EU/mL or EU/mg.

Key Characteristics

• Quantitative endotoxin measurement
• Wide dynamic range
• High sensitivity for low-level detection
• Automated kinetic monitoring improves precision

Advantages Over Gel-Clot

• Provides numeric EU values
• Better suited for trending and stability studies
• Enhanced sensitivity for low-endotoxin materials
• Improved data documentation for technical review

Interference Control

For both methods, inhibition/enhancement testing may be performed to confirm sample matrix does not interfere with the LAL reaction. When glucan interference is suspected, glucan-blocking approaches can be incorporated to improve specificity.

Method Comparison

Sample submission guidance

Recommended information

• Sample form (lyophilized vs solution), concentration (if known), and solvent/buffer identity
• Intended use (research assay, in vivo, regulated non-GMP documentation needs)
• Expected endotoxin limit (if you have one) and desired reporting unit (EU/mL vs EU/mg)
• Any known risk factors for glucan (cellulose filters, plant-derived components, cotton-containing materials)

Handling & contamination control

• Use clean, well-sealed containers; minimize transfers and open-air handling
• Document any filtration steps and materials (filters can be a glucan source)
• Disclose surfactants/detergents, strong solvents, or unusual matrices (can impact assay behavior)
• Submit enough material for dilution series and validity controls (scope-dependent)

References

• USP <85> Bacterial Endotoxins Test
• European Pharmacopoeia 2.6.14 Bacterial Endotoxins
• FDA Guidance for Industry: Pyrogen and Endotoxins Testing
• Williams KL. Endotoxin Detection and Control in Pharma, Limulus Amebocyte Lysate Testing