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.
Figure 5. Pierce Chromogenic Endotoxin Quantitation Kit assay principle. Sample endotoxin activates factor C, which leads to a signal cascade that results in activated protease catalyzing the release of chromogenic pNA. This produces a yellow color that can be measured by absorbance at 405 nm. A standard curve is plotted from the absorbance values generated from a set of diluted endotoxin samples.
Sensitivity and reproducibility
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).
Compatibility
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.
Table 1. Highest acceptable concentration of typical reagents for valid endotoxin spike recovery using the Pierce Chromogenic Endotoxin Quant Kit. Concentrations listed refer to the actual concentration in the sample that produced no decrease in quantitation values when spiked with 0.5 EU endotoxin. Dilutions are expressed in the form of a ratio, where 1:100 means a 100-fold dilution.
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
Product | Unit size | |
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A39552 | 60 reactions | Order now |
A39553 | 240 reactions | Order now |
A39552s | 30 reactions | Order now |