Overview of Endotoxin Testing Methods

Thermo Scientific Pierce Rapid Gel Clot Endotoxin Assay kits provide rapid and easy-to-read qualitative results indicating the presence or absence of gram-negative bacterial endotoxin in a sample. The kits are used for in vitro end-point endotoxin tests and do not require sophisticated equipment or software. They are best suited for samples that are colored and are available in a variety of sensitivities: 0.03 EU/mL, 0.125 EU/mL, 0.25 EU/mL, and 0.5 EU/mL.

Pierce Rapid Gel Clot Endotoxin Assay kits use amebocyte lysates derived from the Limulus horseshoe crab hemolymph to detect endotoxin levels in samples. Limulus amebocyte lysate (LAL) is widely used as a simple and sensitive assay for detection of endotoxin lipopolysaccharide from the membranes of gram-negative bacteria. When endotoxin encounters the amebocyte lysate, a series of enzymatic reactions form a gel-like clot. In a positive test, a clot will form in the sample tube, indicating that the amount of endotoxin in the sample is greater than or equal to the listed sensitivity (in EU/mL) of the kit. A lack of gel clot formation in the tube is considered a negative result indicative of an endotoxin concentration in the test tube below the kit’s sensitivity (Figure 3).

NEW Pierce Rapid Gel Clot Endotoxin Assay Kit

The Thermo Scientific Pierce Chromogenic Endotoxin Quantitation Kit accurately measures endotoxins at levels as low as 0.01 EU/mL in samples. This kit is an endpoint amebocyte lysate assay that accurately detects and quantitates endotoxins (lipopolysaccharides) in a variety of sample types, including proteins, peptides, nucleic acids, and antibodies.

The principle of the assay is based on the activation of factor C, factor B, and pro–clotting enzyme in the amebocyte lysate in the presence of endotoxin. The amount of endotoxin is quantitated by the addition of a chromogenic substrate, Ac-Ile-Glu-Ala-Arg-pNA. The endotoxin-activated pro–clotting enzyme catalyzes the release of p-nitroaniline (pNA) to produce a yellow color (Figure 4).

Figure 4. Coagulation cascade in horseshoe crab blood. LPS (endotoxin) activates plasma membrane–bound factor C. The activated factor C activates factor B, which activates the clotting enzyme that triggers the exocytotic release of the clotting cascade. Upon addition of a chromogenic substrate, Ac-Ile-Glu-Ala-Arg-pNA, the activated protease, clotting enzyme, catalyzes the release of p-nitroaniline (pNA), resulting in a yellow color that can be quantitated by measuring the absorbance at 405 nm and extrapolating from a standard curve.

After the reaction is stopped, the released pNA is photometrically measured at 405 nm (Figure 5). The developed color intensity is directly proportional to the amount of endotoxin present in the sample and is calculated using a standard curve.

Sensitive endotoxin testing is essential because of the sample limitations and low endotoxin levels required for cell culture and animal research. The Pierce Chromogenic Endotoxin Quant Kit offers high-sensitivity and reproducibility with two linear dynamic ranges of 0.01–0.1 EU/mL and 0.1–1.0 EU/mL (Figure 6). The test-to-test and operator-to-operator reproducibility of this endpoint amebocyte lysate assay resulted in a coefficient of variation (CV) of 3%. The lower sensitivity range permits higher dilution of samples that are limited in quantity or contain interfering substances, therefore helping to avoid potential false negatives or false positives in endotoxin quantitation.

Figure 6. Standard curves for the Pierce Chromogenic Endotoxin Quantitation Kit. The standard curves show exceptional linearity with r2 = 0.99. The Pierce Chromogenic Endotoxin Quant Kit has a lower-range standard curve of 0.01–0.1 EU/mL. The standard curve of 0.1–1.0 EU/mL represents a 14-minute incubation with endotoxin standards and amebocyte lysate, followed by a 6-minute incubation with the chromogenic substrate. For the lower range, a 30-minute incubation with endotoxin standards and amebocyte lysate was followed by a 6-minute incubation with the chromogenic substrate. Consistency and reproducibility (n = 17; CV = 3%) is shown for the low-range standards (0.01–0.1 EU/mL).

Amebocyte lysate assays can be affected by many factors that can cause inhibition or enhancement leading to false negatives or false positives. Factors that can lead to inhibition of the amebocyte lysate assay include reaction temperature, sample pH, ionic strength, and metal ions (e.g., magnesium and calcium). Serum proteins, nucleic and fatty acids, surfactants, and chelating reagents (e.g., EDTA and heparin) cause changes in molecular structure of endotoxin aggregates that can result in inaccurate or total inhibition of the amebocyte lysate assay. In addition, surfactants (e.g., Triton X-100, SDS, deoxycholate) used in various protein workflows alter the supramolecular structure of lipopolysaccharides and therefore can interfere with their detection and quantitation. If any of these interfering substances are present in test samples, the samples need to be diluted.

Table 1 summarizes compatibility levels of common reagents with the amebocyte lysate assay. To be considered free of interfering factors in the test, the measured concentration of endotoxin added to the sample must be within 50%–200% of the known added amount.

Another common interfering substance in the amebocyte lysate assay is (1,3)-β-D-glucan, a cell wall component of bacteria and fungi. Although β-glucan is not pyrogenic, it can activate factor G, which is able to trigger the coagulation cascade, producing false positives in the assay (Figure 7). The Pierce Chromogenic Endotoxin Quant Kit is compatible with β-glucans, and in samples containing ≤10 ng/mL of (1,3)-β-D-glucan, no enhancement is exhibited.

Figure 7. Activation of the clotting enzyme by (1,3)-β-D-glucan. The presence of β-glucans activates a pathway that is independent of the amebocyte lysate response to endotoxins resulting in false-positive determination of bacterial endotoxins in the sample. The Pierce Chromogenic Endotoxin Quant Kit is resistant to β-glucans. Red indicates inactive enzymes, and green indicates active enzymes.

Pierce Chromogenic Endotoxin Quant Kit

Endotoxin-contaminated protein or antibody samples transfected into cells or injected into an animal host can initiate a strong immune response, resulting in systemic inflammatory response syndrome (SIRS) and/or sepsis. Elimination of endotoxins from samples produced from gram-negative bacteria prior to cell transfection or animal injection is a necessity. Ultrafiltration, polymixin B affinity resin, or resin- or membrane-based chromatography are the traditional methods of endotoxin removal. These methods have limitations in protein recovery or endotoxin binding capacity or have toxicity concerns.

The Pierce High Capacity Endotoxin Removal Resin is a modified ε-poly-L-lysine [poly (ε-lysine)] affinity resin (nontoxic polymer of the natural amino acid lysine) that displays both excellent endotoxin binding capacity and protein recovery. It is effective in eliminating endotoxins from samples containing proteins of various sources, sizes, and charges. The high binding capacity and low protein retention of this resin make it suitable for many protein sample types, including antibodies.

Figure 8 shows how the poly (ε-lysine) affinity ligands on the Pierce High Capacity Endotoxin Removal Resin bind endotoxins through both cationic and hydrophobic interactions.

The Pierce High Capacity Endotoxin Removal Resin has a 2,000,000 EU/mL binding capacity, enabling endotoxin levels to be reduced by 99% in samples containing initial endotoxin levels of 10,000 EU/mL. Typical protein samples processed with the Thermo Scientific Pierce High Capacity Endotoxin Removal Resin have a final endotoxin concentration below 5 EU/mL (Figure 9).

Pierce High Capacity Endotoxin Removal Resin

The Invitrogen Qubit Endotoxin Detection Assay Kit and the Invitrogen Quant‑iT Endotoxin Detection Assay Kit, designed for use with the Invitrogen Qubit Flex Fluorometer and fluorescence microplate readers, respectively, offer efficient, fluorescent endpoint assays that quantify endotoxins in various sample types using a streamlined workflow.

The Qubit Endotoxin Detection Assay, designed for use with Qubit Flex Fluorometers, is an efficient, fluorescent endpoint assay that uses amebocyte lysates to quantify endotoxin in various sample types such as protein, peptides, antibodies or nucleic acid samples. The assay can be run with variable sample volumes and offers a dynamic range from 0.01-10.0 EU/mL using a streamlined workflow (Figure 10).

Key features and benefits of the Qubit Endotoxin Detection Assay Kit include:

• High sensitivity and broad range - Detect as little as 0.01 EU/mL to 10.0 EU/mL
• Versatility – Suitable for a wide range of samples, including proteins, plasmid preparations, DNA and RNA
• Easy-to-use – When paired with the Qubit Flex Fluorometer, calculations are performed automatically, reducing the potential for error

NEW Invitrogen Quant-iT™ Endotoxin Detection Assay Kit
The Quant-iT Endotoxin Detection Assay, designed for use with fluorescence microplate readers, offers an efficient, fluorescent endpoint assay that uses amebocyte lysates to quantify endotoxin in various sample types such as protein, peptides, antibodies or nucleic acid samples. The assay can be performed on variable sample volumes, and offers a dynamic range of 0.01–10.0 EU/mL using a streamlined workflow (Figure 11).