The Thermo Scientific Pierce LAL Chromogenic Endotoxin Quantitation Kit measures the amount of endotoxin in a protein, peptide or antibody sample using the Limulus Amebocyte Lysate (LAL) assay.
The endotoxin concentration in a sample is measured using the Pierce LAL Chromogenic Endotoxin Quantitation Kit via a chromogenic signal generated in the presence of endotoxins. Samples can be measured on a microplate absorbance reader at 405nm. A standard curve is created using the E. coli endotoxin standard included with each kit to calculate endotoxin levels as low as 0.1 EU/mL, where one endotoxin unit/mL (EU/mL) equals approximately 0.1ng endotoxin/mL of solution. Protein and antibody samples can be assayed in about 30 minutes. Determining endotoxin levels is important to assess the efficiency of endotoxin removal methods and prevent endotoxic shock, inflammation and/or sepsis in tissue culture cells and animals injected with endotoxin contaminated proteins.
- Sensitive – detect as little as 0.1 EU/mL (approx. 0.01ng endotoxin per mL)
- Fast – perform this assay in 30 minutes using a 96-well microplate
- Economical – assay requires only 10μL of a protein sample
- Accurate – E. coli O111:B4 standard in each kit enables accurate endotoxin quantitation
- Versatile – 405nm absorbance reading is compatible with common ELISA plate readers
Kit contains Limulus Amebocyte Lysate (LAL), E. coli endotoxin standard, chromogenic substrate and endotoxin-free water to prepare assay reagents.
Quantitation of endotoxin levels in a protein, peptide or antibody solution
The LAL method for measuring endotoxin is based on the interaction of endotoxins with the proenzyme Factor C found in circulating amebocytes of the horseshoe crab Limulus polyphemus. The proteolytic activity of this proenzyme is activated in the presence of lipopolysaccharides (endotoxins) derived from the outer cell membrane of gram-negative bacteria such as E. coli. The Chromogenic Limulus Amebocyte Lysate assay measures endotoxin levels by measuring the activity of this protease in the presence of a synthetic peptide substrate that releases p-nitroaniline (pNA) after proteolysis, producing a yellow color that can be measured by reading the absorbance at 405nm.
To accurately measure endotoxin levels in a sample, the LAL assay uses an endotoxin standard of known concentration that is derived from E. coli strain O111:B4. This standard is provided with each kit and is used to create a standard curve. The endotoxin concentration is determined by extrapolating the absorbance of an unknown sample against this standard curve, similar to ELISA or total protein quantitation assays.
|Pierce LAL Chromogenic Endotoxin Quantitation Kit reaction scheme. A small volume of the sample (10μL) is combined with the Limulus Amebocyte Lysate, and endotoxins in the sample activate the proteolytic activity of Factor C. When the chromogenic substrate is added, the activated protease catalyzes the cleavage of p-nitroalinine (pNA), resulting in yellow color that can be quantitated by measuring the absorbance at 405nm (A405) and extrapolating against a standard curve.
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