FDA-licensed LAL Reagents

Limulus amebocyte lysate (LAL) is an aqueous extract of blood cells (amebocytes) from the Atlantic horseshoe crab, Limulus polyphemus. Comprised of proteins, LAL assay is used to detect the presence of endotoxins, a cell wall component of Gram-negative bacteria that causes a pyrogenic response (fever) and symptoms of septic shock. Even if dead, these components trigger a response in LAL, causing it to clot and coagulate.

The most expensive LAL assay is the one that must be repeated because of invalidity. The generation of valid results for bacterial endotoxins testing (BET) necessitates robust test procedures, well-maintained equipment, and properly trained personnel, but too often it is cost, not quality, that is the driving force in the BET decision-making process.

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and our FDA-Licensed LAL Reagents?

Lab technician examining cartridge that is used for endotoxin detection assaysCharles River offers a complete portfolio of reagents and accessories to aid and ensure bacterial endotoxin-free products during manufacturing. 

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Charles River has developed and optimized a range of quantitative and qualitative LAL reagent water formulations that provides increased sensitivity, greater linearity, and superior interference resistance. Our LAL assays and reagents are licensed by the FDA for product release, and all of our accessory products are certified for the appropriate LAL testing requirements. Our LAL reagent water delivers extreme precision and reliability, minimizing invalid results and the need to retest.
 

Lab technician using nexgen-pts to run endotoxin detection assay with the innovative Charles River Endosafe cartridge technology

Endosafe® Cartridge Technology: The Endosafe® cartridge technology is our advanced solution to our customers’ need for higher sensitivity in the LAL assay and faster quantitative results. Created to improve and refine our use of LAL, the cartridge technology cuts out a significant amount of the raw material and accessories required for traditional LAL assay methods while reducing time-consuming preparation and technician variability.

 

lab technician performing kinetic chromogenic LAL test using BioTek® incubating microplate reader

Kinetic Chromogenic LAL: A fully quantitative and uniquely stable kinetic chromogenic LAL reagent, Endochrome-K™ includes an enhanced KCA formulation that delivers an unrivaled combination of sensitivity, linearity, and interference resistance. Our Endochrome-K™ LAL  reagent offers quantitative endotoxin values in roughly an hour, depending on the user’s desired sensitivity, and the stability of the reconstituted LAL allows for extended bench time and reuse, making it a highly economical solution.

 

Vials of kinetic turbidimetric reagent for LAL endotoxin detection assay

Kinetic Turbidimetric LAL: Our kinetic turbidimetric (KTA) LAL reagents allow you to improve your testing with a single FDA-licensed product that performs both kinetic and gel-clot analysis with accelerated reaction times and no pre-incubation necessary. Our second-generation reagent, KTA2, releases one of the fastest turbidimetric LAL reaction times in the industry, and our enhanced LAL reagent water formulations deliver an unrivaled combination of sensitivity, linearity, and interference resistance properties with minimal assay times.

 

Results of a Gel-clot assay performed by a lab technician

Gel-Clot LAL: The gel-clot LAL assay is a basic qualitative method best used for low-volume laboratories. Our lysate features a firm gel over a wide range of sensitivities as the buffered reagent provides improved interference resistance for routine LAL assays. Besides our reagents, we carry a host of LAL Assay Accessories. An integral part of endotoxin testing is the selection of LAL test accessories that facilitate data collection free of artifacts and sources of interference. The selection of non-interfering accessories for a BET is not only a pharmacopeial directive but also a regulatory expectation.

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Frequently Asked Questions (FAQs) About LAL Assays and LAL Reagents

  • How does the LAL assay work?

    The bacterial endotoxin test uses Limulus amoebocyte lysate (LAL), which is an aqueous extract derived from horseshoe crab blood. The LAL will either become opaque, or in the case of kinetic chromogenic lysate, yellow, in the presence of endotoxin. Endotoxin is a fever-producing byproduct of Gram-negative bacteria.

  • What is the LAL assay used for?

    LAL assays are used to detect and measure instances of bacterial endotoxin contamination in drugs, biological products, medical devices, and others. The LAL assay is the most sensitive and specific means to detect bacterial endotoxin.

  • When should an LAL assay be performed?

    The LAL assay should be performed on raw materials and final products for intravenous intrathecal drug products, in addition to medical devices. Performing LAL assays provides data on potentially contaminated products, in which the contaminants would directly enter the bloodstream of the person to which it is administered. Bacterial endotoxins cause a pyrogenic reaction that can be deadly in many cases.

  • What is LAL reagent water?

    LAL reagent water is water for injection (WFI) that is endotoxin-free, which is mainly used in the BET to reconstitute LAL and controlled standard endotoxin (CSE), and to make sample dilutions.

  • Which is the best LAL assay for endotoxin testing?

    The gel-clot LAL assay is very sensitive and accurate, however it is a time-consuming process as each assay takes 30-40 minutes of an analyst’s time. And as there is no automation for this method, there is potential for bias or human error when the technician reads and interprets the test results.

    The chromogenic LAL assay is user-friendly and can be automated, which decreases the amount of time required to perform the test, allows more tests to be completed per unit of time, and calculations can be performed simply. Kinetic turbidimetric and chromogenic LAL assays both give a quantitative result. They require, however, more technician time and preparation, resulting in room for human error.

    Endosafe cartridges eliminate the need for a standard curve, reducing the amount of time to result and decreasing the chance for human error. Each lab needs to decide for themselves which LAL assay works best for them based on throughput, human capital, money and time resources, data integrity strategy, and sustainability practices. 

  • What are the methods for bacterial endotoxin testing (BET)?

    The main methods used for bacterial endotoxin testing use Limulus Amoebocyte Lysate (LAL) and are gel-clot, kinetic chromogenic, and kinetic turbidimetric. The gel-clot method is a qualitative BET and is used to detect the presence or absence of endotoxin in the prepared sample. When endotoxin encounters the LAL reagent, it triggers a series of enzymatic reactions. The enzymes change the amoebocyte coagulogen present in LAL reagent to form a detectable gel-clot.

    After the test is performed, if a clot has formed and remains intact at the bottom of the tube, the result of the test is positive, meaning that there is detectable endotoxin in the sample. When no clot forms or the clot dissipates when the tube is turned upside down, the result is negative, meaning there is no detectable endotoxin in the sample based on the sensitivity of the LAL reagent.

    The kinetic chromogenic method measures the absorbance of light in LAL reagent to determine if endotoxin is present by visible color changes. The degree of color and the rate at which the color appears is directly proportional to the amount of endotoxin within the sample.

    The chromogenic method is user-friendly and can be automated, which decreases the amount of time required to perform the test, allows more LAL assays to be completed per unit of time and calculations can be performed simply. Charles River Endosafe® cartridge technology uses the chromogenic method.

    The kinetic turbidimetric method is a modified extension of the gel-clot test. It measures the change in turbidity over time. The LAL reagent is altered so it is unable to form a solid clot when exposed to endotoxin. The amount of endotoxin present is inversely proportional to the reaction time.