What endotoxins are
Endotoxins are lipopolysaccharides (LPS), large molecules that make up much of the outer membrane of Gram-negative bacteria such as Escherichia coli. They are released when bacteria grow, divide or die, and they are very stable: normal autoclaving does not destroy them, freeze-drying does not remove them, and they pass through the 0.2 µm filters used to remove bacteria. A material can therefore be sterile and still contain endotoxin.
In the laboratory, endotoxin matters because very small amounts activate immune cells such as macrophages, mainly through the TLR4 receptor. In cell culture and other work with biological systems, endotoxin carried in with a reagent can change results that are then wrongly attributed to the material under study.
Where endotoxin comes from
Because drying does not remove endotoxin, the level in a lyophilized powder reflects what was present in the solution before freeze-drying. Endotoxin enters with anything that has been in contact with bacteria:
- Water used in synthesis, purification or rinsing.
- Buffers, salts and other reagents.
- Glassware, tubing and plastics that are not made endotoxin-free.
- Handling during filling and packing.
How the LAL test works
The standard method is the Limulus amebocyte lysate (LAL) test. Its reagent is made from blood cells of the horseshoe crab, whose clotting system is triggered by endotoxin: endotoxin activates factor C, which starts an enzyme cascade that ends in a clot. The test versions differ in how they read that reaction.
Each run includes endotoxin standards and a check that the sample itself does not inhibit or enhance the reaction, usually by adding a known amount of endotoxin to the sample and measuring how much is recovered. Peptides, metal ions and extreme pH can interfere, so laboratories often dilute a sample until it no longer does.
| Method | What is read | Type of result |
|---|---|---|
| Gel-clot | Whether a firm gel forms after incubation. | Above or below the reagent’s stated sensitivity; a dilution series gives an end-point value. |
| Kinetic turbidimetric | How fast the mixture becomes cloudy. | A value read from a standard curve. |
| Chromogenic | Yellow colour released from a synthetic substrate. | A value read from a standard curve. |
| Recombinant factor C (rFC) | Fluorescence from a substrate cut by recombinant factor C. | A value read from a standard curve, without horseshoe crab blood. |
Endotoxin units: EU per vial, per mg and per mL
Results are given in endotoxin units (EU), a measure of activity against a reference endotoxin; for the usual reference standard, 1 EU is roughly 0.1 to 0.2 ng of endotoxin. A result can be stated per vial, per milligram of material or per millilitre of solution. To compare a result per vial with a limit per milligram, divide it by the amount of material in the vial, using the measured content if the report gives one.
A result below the lowest point of the standard curve is reported as less than that value, for example < 0.005 EU/mL, not as zero. There is no single endotoxin limit for research materials: compare the result with the limit your own protocol or cell system needs, expressed in the same unit.
The endotoxin results on our reports
For example, the reports below in our report library include an endotoxin result. Each gives the result per vial and a measured amount of material, so a value per milligram can be worked out.
These reports give the endotoxin result without a specification next to it, so the result is a measurement rather than a pass against a limit. The per-mg column in the table is our own arithmetic from the two printed values; it is not printed on the reports. If your work depends on the method, ask the issuer which one was used.
The issuer (TJQXGD) states that the data come from a third-party partner laboratory, that neither it nor the laboratory holds CMA/CNAS accreditation, and that this document has no legal certification validity. Each report covers only the sample and batch printed on it. Verify it on the issuer’s website and check that the lot number matches the goods you receive.
| Report | Endotoxin, as printed | Amount, as printed | Per mg (our calculation) |
|---|---|---|---|
| SNAP-8 10 mg, NP8 10-20260914S8218 | 0.259 EU/vial | Net content 10.85 mg | about 0.024 EU/mg |
| NAD+ 1000 mg, NAD1000-20260914S8218 | 1.156 EU/Vial | Net content 1034.21 mg | about 0.0011 EU/mg |
| Glutathione 1500 mg, GTT1500-20260907S7260 | 1.037 EU/Vial | Peptide weight 1464.81 mg | about 0.00071 EU/mg |
What to ask for when endotoxin matters
Ask for the documents for the exact lot in your quote. A report describes only the batch it tested, so check that its lot number matches the lot you receive.
- The endotoxin result for the exact lot you receive, with its unit.
- The method (gel-clot, turbidimetric, chromogenic or recombinant factor C) and its sensitivity or standard-curve range.
- Whether the inhibition and enhancement check passed.
- The limit your protocol needs, in the same unit as the result.
- Endotoxin-free water, glassware and plastics for your own work, so that endotoxin is not added after receipt.
For laboratory research. Not for human or animal use.