Endotoxin contamination is a common problem with recombinant proteins and nucleic acids purified from gram-negative bacteria such as E. coli. Endotoxins are lipopolysaccharides (LPS), which are heat-stable molecules associated with the outer membranes of certain gram-negative bacteria. When bacterial cells are actively growing or when their membranes disintegrate upon death, the essential LPS components of the cell wall are released into the surrounding environment. Lipopolysaccharides are large molecules ranging in size from 3,000 to 4,000 Da, consisting of a hydrophobic lipid group covalently bound to a long complex polysaccharide tail (Figure 1). Endotoxin contamination is dangerous and can trigger endotoxic shock, inflammation, or sepsis in animals and tissue culture.

Introduction

Endotoxin contamination is a common problem with recombinant proteins and nucleic acids purified from gram-negative bacteria such as E. coli. Endotoxins are lipopolysaccharides (LPS), which are heat-stable molecules associated with the outer membranes of certain gram-negative bacteria. When bacterial cells are actively growing or when their membranes disintegrate upon death, the essential LPS components of the cell wall are released into the surrounding environment. Lipopolysaccharides are large molecules ranging in size from 3,000 to 4,000 Da, consisting of a hydrophobic lipid group covalently bound to a long complex polysaccharide tail (Figure 1). Endotoxin contamination is dangerous and can trigger endotoxic shock, inflammation, or sepsis in animals and tissue culture.
Thermo Fisher Scientific has extended its endotoxin detection line to include the Thermo Scientific™ Pierce™ Chromogenic Endotoxin Quant Kit, which accurately detects endotoxins at levels as low as 0.01 EU/mL in samples. The Pierce Chromogenic Endotoxin Quant 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.

How does the assay work?

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 1). After the reaction is stopped, the released pNA is photometrically measured at 405 nm (Figure 2). The developed color intensity is directly proportional to the amount of endotoxin present in the sample and is calculated using a standard curve. Here we present data demonstrating the performance of Pierce endotoxin quantitation kits.

Figure 1. 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. Red indicates inactive enzymes, and green indicates active enzymes.

Figure 2. Assay protocol for Pierce endotoxin quantitation kits.

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.

Download and read the white paper to learn more about the Pierce Chromogenic Endotoxin Quant Kit.