Overview of Endotoxin Testing Methods - US (2024)

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.

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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).

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.