Endotoxin Testing

Endotoxins, also known as lipopolysaccharides (LPS), are toxic substances found in the outer membrane of Gram-negative bacteria. These endotoxins are released into the environment when bacterial cells die or undergo lysis [1].

History of Endotoxin Testing

Endotoxin testing has evolved over the years, starting from pyrogen testing to more refined methods like gel clot, turbidimetric, or quantitative chromogenic Limulus Amebocyte Lysate (LAL)-based assays. These lysates are prepared from the amebocytes of the horseshoe crab, producing a clot or gel upon exposure to bacterial endotoxin [2]. Pyrogen testing, which began in the early 1900s, aimed to detect fever-inducing substances in parenteral medicines, often derived from microorganisms. The most potent pyrogens are bacterial endotoxins, which are a subset of pyrogens [3].

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Image 1: Endotoxin Assay at Boston BioProducts. In our quality control lab, we have optimized the efficiency in which products may be tested for endotoxin, ensuring that necessary endotoxin specifications are met in a time efficient manner. Here, a 96-well plate is carefully prepared for an endotoxin assay.

Importance of Endotoxin Testing

Testing for bacterial endotoxins is crucial because their presence in buffers, reagents, or solutions can lead to various experimental issues, and contaminated products can be harmful to human and living models. In humans, pyrogens entering the bloodstream may initiate secretion of pro-inflammatory cytokines by monocytes. When high concentrations of pyrogens enter the blood stream they cause fever, chills, septic shock and in severe cases even death [4]. Common sources of endotoxins in these material include the water used, raw materials, equipment, and packaging components. Controlling microbiological and endotoxin contamination in these sources is a fundamental part of good manufacturing practice.

What test is used at Boston BioProducts?

Boston BioProducts employs a highly sensitive colorimetric-based method using Toxin Sensor Chromogenic LAL Endotoxin Assay Kits. This assay detects and quantifies endotoxins using Limulus Amebocyte Lysate (LAL). In this method, endotoxins present in samples catalyze the proteolytic activation of pro-enzymes in LAL. The activated enzyme leads to the cleavage of a colorless substrate and the production of a colored product. This colored result is quantitatively measured at 545 nm using a microplate reader.

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Images 2 & 3: Loading and Analysis of 96-well plate endotoxin assay. This process involves careful execution and examination of data. Even the slightest contamination of endotoxin can cause an assay to fail.

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Image 4: Results of a successful endotoxin assay should yield an R2 value of 1. This value represents the changes in dilutions used, as these dilutions are created to support a standard curve under the formula Y=A*X+B. An R2 value here is a measure of how well our statistical model fits the derived data. It ranges from 0 to 1, where 0 means that the model explains none of the variability of the data, and 1 means that the model explains all the variability of the data.

Considerations and Limitations of Endotoxin Testing
Level of Endotoxin in Water Ensure that the water used as a solvent has an extremely low endotoxin level, and regularly monitor and maintain the water filtration system used for development of products. In manufacturing situations lacking access to such filtration systems, it is most cost efficient and safe to purchase water tested for endotoxin.
Cleanliness of the Environment and Containers Prepare buffers in a clean environment using clean containers and filter them immediately after preparation to prevent microbial contamination.
pH Adjust the pH of the solution to fall within the 6.4 to 8 pH range, as the endotoxin assay functions best in this range.
Ionic Strength High salt concentration (> 1M) can inhibit the endotoxin assay, so samples with high salt concentrations should be diluted.
Presence of Detergents Detergents can interfere with the detection of endotoxins, so buffer solutions with detergents should be diluted before testing.
Presence of Chelators Chelators like EDTA, EGTA or Citrate can inhibit the endotoxin assay, so dilution of samples with chelators is necessary.
Presence of Chaotropic Agents Chaotropic agents like urea or guanidine chloride can alter endotoxin aggregates that result in inhibition of LAL activity, so appropriate dilutions are required.
Presence of Organic Solvents High concentrations of organic solvents can dissolve lipid A, the lipid portion of endotoxins, leading to inhibition of the assay.
Presence of Sugars Impure sugars containing β-glucans can yield false-positive results, so use highly purified sugars.
Presence of Serum Proteins Serum proteins such as Bovine Serum Albumin (BSA) or Human Serum Albumin (HSA) can affect endotoxin detection, so dilute samples containing serum albumin.
Presence of DNA and Fatty Acids Nucleic acids and fatty acids can cause inaccurate results, so monitor and correct these parameters.
Physical Agents Viscosity, color, and turbidity of samples can interfere with absorbance measurements, so manipulate these parameters before testing.

In addition to Endotoxin Testing, Boston BioProducts provides a comprehensive set of QC tests for custom reagents. Learn more about custom reagent development services.

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