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class="content"><article class="post-3300 post type-post status-publish format-standard has-post-thumbnail category-connect-to-science category-general entry" aria-label="Endotoxins 101: Guide to Bacterial LAL Endotoxin Testing"><header class="entry-header"><h1 class="entry-title">Endotoxins 101: Guide to Bacterial LAL Endotoxin Testing</h1> <span class="post-info-author"> By <a href="https://www.thermofisher.com/blog/author/danadamico/" title="Posts by Dana D'Amico" rel="author">Dana D'Amico</a> </span> <span class="post-info-date"> 09.27.2024 </span> </header><div class="entry-content"><p><img decoding="async" class="aligncenter size-large wp-image-3354" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-7-1024x576.jpg" alt="" width="760" height="428" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-7-1024x576.jpg 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-7-300x169.jpg 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-7-768x432.jpg 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/Child_Top-Pod-7.jpg 1280w" sizes="(max-width: 760px) 100vw, 760px" /></p> <p> </p> <h2>Understand the different LAL endotoxin testing options, and learn tips to avoid common workflow challenges.</h2> <p> </p> <h3><strong>Table of Contents </strong></h3> <ul> <li><a href="#what">What are endotoxins?</a></li> <li><a href="#why">Why is bacterial endotoxin testing (BET) important?</a></li> <li><a href="#types">What type of bacterial endotoxin testing (BET) should I use?</a> <ul> <li><a href="#LAL">LAL test</a></li> <li><a href="#qubit">Qubit™ and Quant-iT™ Endotoxin Detection Assays</a></li> <li><a href="#recombinant">Recombinant endotoxin assays</a></li> </ul> </li> <li><a href="#challenges">What challenges and tips should I consider during endotoxin testing?</a> <ul> <li><a href="#contamination">Contamination protocol</a></li> <li><a href="#controlstandards">Control standards and calibration</a></li> <li><a href="#dataprocessing">Data processing</a></li> <li><a href="#training">Training</a></li> </ul> </li> <li><a href="#learnmore">Where can I learn more about emerging endotoxin testing methods?</a></li> </ul> <p> </p> <h3 id="what">What are endotoxins?</h3> <p>Endotoxins, or lipopolysaccharides (LPS), are the structural glycolipids that envelop and protect gram-negative bacteria like <em>E. coli, </em>the world’s most widely studied prokaryotic model<em>. </em>Gram-negative bacteria shed endotoxins throughout their life cycle – but particularly during lytic death where they can release large amounts at once.</p> <p>Endotoxins have a powerful activating effect on animal immune systems. Large release events can trigger shock, fever, inflammation, and sepsis. Endotoxins are also the most common pyrogens found in medical devices, and regulators set standards for amount of endotoxin permitted. Endotoxin testing is just one of several types of pyrogen tests necessary in the food and healthcare space.</p> <div id="attachment_3355" style="width: 799px" class="wp-caption aligncenter"><img decoding="async" aria-describedby="caption-attachment-3355" class="size-full wp-image-3355" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/endotoxins.png" alt="Endotoxin molecular structure" width="789" height="296" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/endotoxins.png 789w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/endotoxins-300x113.png 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/endotoxins-768x288.png 768w" sizes="(max-width: 789px) 100vw, 789px" /><p id="caption-attachment-3355" class="wp-caption-text"><strong>Structure of bacterial endotoxin.</strong> Endotoxins are complex lipopolysaccharides and are biologically active structural components of the outer cell membrane of gram-negative bacteria. They consist of a core oligosaccharide chain, O-specific polysaccharide side chain (O-antigen), and a lipid component, lipid A, which is responsible for the toxic effects. In the early 1950s, Frederick Bang discovered that the blood cells (amebocytes) of the horseshoe crab clot in the presence of low levels of endotoxins. The gel-clot method established from this observation is an economical, specific, and sensitive method to detect endotoxins. The use of the amebocyte lysate has become the industry standard for endotoxin detection in the pharmaceutical and food industries as well as life science and medical research.</p></div> <p> </p> <h3 id="why"><strong>Why is bacterial endotoxin testing (BET) important?</strong></h3> <p>Because of their medical significance <em>in vivo</em> and ability to interfere with the efficiency of many common <em>in vitro</em> research processes like cell culture and transfection, endotoxin monitoring is a must in the life sciences.</p> <p>A few scenarios where understanding endotoxin levels is key:</p> <ul> <li>For biological sample processing in the manufacture of therapeutic proteins or cell and gene therapies, to prevent downstream contamination</li> <li>When developing liquid nanoparticle (LNP) or adeno-associated (AAV) mRNA delivery vehicles for living models</li> <li>During biopharmaceutical production, to adhere to endotoxin limit regulations and ensure patient safety and product quality</li> <li>When purifying plasmids and transfecting endotoxin-sensitive cell lines, to preserve cell health and transfection efficiency</li> <li>When verifying water potability and the safety of commercial food products</li> </ul> <p> </p> <h3 id="types"><strong>What type of bacterial endotoxin testing (BET) should I use?</strong></h3> <p>Researchers today have a handful of options for bacterial endotoxin testing (BET), each with their own pros and cons with respect to ease-of-use, sensitivity, cost, and regulatory considerations.</p> <ul> <li id="LAL">The<strong> Limulus amebocyte lysate, </strong>or<strong> LAL endotoxin test, </strong>is by far the most common assay used for endotoxin detection. LAL is extracted from the blood cells of the Atlantic horseshoe crab, <em>Limulus polyphemus. </em>Some assay formulations may use a similar lysate (TAL) extracted from the Asian <em>Tachypleus </em>species of crabs. Amebocyte lysates are used in simple qualitative gel-clot tests which can provide a quick positive (clot) or negative (no clot) to detect the presence of endotoxins. LAL and TAL tests can also provide quantitative information via chromogenic methods (color-based) or turbidimetric (turbidity-based) analysis. The United States Pharmacopeia (USP) has developed a harmonized standard for LAL endotoxin testing.</li> </ul> <div id="attachment_3356" style="width: 770px" class="wp-caption aligncenter"><img loading="lazy" decoding="async" aria-describedby="caption-attachment-3356" class="size-large wp-image-3356" src="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/LAL-1024x972.png" alt="Endotoxin gel clot reaction (LAL test)" width="760" height="721" srcset="https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/LAL-1024x972.png 1024w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/LAL-300x285.png 300w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/LAL-768x729.png 768w, https://www.thermofisher.com/blog/life-in-the-lab/wp-content/uploads/sites/10/2024/09/LAL.png 1054w" sizes="(max-width: 760px) 100vw, 760px" /><p id="caption-attachment-3356" class="wp-caption-text"><strong>Endotoxin gel clot reaction.</strong> LAL clots in the presence of endotoxin-containing sample via an enzymatic cascade reaction. If endotoxin is not present in the sample, no clotting occurs.</p></div> <p> </p> <ul> <li id="qubit"><strong>Qubit™ and Quant-iT™ Endotoxin Detection Assays </strong>are a subset of amebocyte lysate-based assays which use fluorescence to provide an easy-to-use workflow and sensitive dynamic range. The end-point assay can detect 0.01 EU/mL to 10.0 EU/mL depending on the sample input volume. This accommodates many regulatory endotoxin limits for <em>in vivo </em>studies and products which can be as low as 0.1 EU/mL. Qubit and Quant-iT assays can be used with protein, antibody, and nucleic acid sample types. When paired with the Qubit Flex Fluorometer, calculations are performed automatically and the guided workflows can help to reduce error. Together, the Qubit and Quant-iT Endotoxin Detection Assays provide results comparable to other BET methods and can be validated to comply with Pharmacopeia standards.</li> </ul> <ul> <li id="recombinant"><strong>Recombinant assays</strong> sidestep the use of animal-derived components by using a recombinant protein substitute for LAL. While this process typically follows a similar process to the LAL assay, some recombinant workflows can require lengthy incubation times or present reduced dynamic ranges for endotoxin detection. Additionally, recombinant assays often require additional validation as alternative methods for use in regulated workflows.</li> </ul> <h5><a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/endotoxin-quantitation-removal/overview-of-endotoxin-testing-methods.html" target="_blank" rel="noopener">Learn more about different assay types >></a></h5> <p> </p> <p><iframe loading="lazy" title="Invitrogen Quant-iT Endotoxin Detection Assay Kit" width="760" height="428" src="https://www.youtube.com/embed/HW3drv1ZAVo?feature=oembed" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" referrerpolicy="strict-origin-when-cross-origin" allowfullscreen></iframe></p> <h3></h3> <h3><strong>What challenges and tips should I consider during endotoxin testing?</strong></h3> <p> </p> <h4 id="contamination"><strong>Contamination Protocol </strong></h4> <p>Few things in the lab can feel as deflating as cross-contamination, and it’s no wonder that so many struggle with unwanted endotoxins – the LPS molecular structure itself has stacked the deck with a hydrophobic Lipid A anchor that easily adsorbs to the plastic (and occasionally, glass) surfaces of common labware. Likewise, these amphiphilic LPS molecules are extraordinarily heat-stable, far beyond the limits of standard lab sterilization processes.</p> <p>Still, there are a few things that you can control to help reduce cross-contamination, including:</p> <ul> <li>Using endotoxin-free LAL <a href="https://www.thermofisher.com/order/catalog/product/Q32891" target="_blank" rel="noopener">reagents</a>, <a href="https://www.thermofisher.com/search/results?query=pierce%20pyrogen%20free&focusarea=Search%20All" target="_blank" rel="noopener">glassware</a>, and <a href="https://www.thermofisher.com/order/catalog/product/Q32893" target="_blank" rel="noopener">plasticware,</a> remembering that many products labelled as “sterile” might not be endotoxin-free</li> <li>Examining your handling procedures to minimize touchpoints and change gloves often</li> <li>Staying aware of the detection limits of your consumables and how they compare to your detection range needs, as some cannot test at lower ranges</li> </ul> <h4 id="controlstandards"><strong>Control Standards and Calibration </strong></h4> <p>Standards provide a known concentration of endotoxins for accurate calibration and measurement of sample values. Here are a few tips:</p> <ul> <li><strong>Endotoxin standards:</strong> You should use certified standards to generate a standard curve – USP guidelines recommend three standards and a blank to be run in replicates. This recommendation would cover a 100X dynamic range, and you should add an additional standard for every 10X addition.</li> <li><strong>Spike recovery tests: </strong>Perform spike recovery tests to ensure that the assay can accurately detect endotoxin in your specific sample matrix. USP85 suggests between 50-200% recovery, though more rigorous labs may want between 75-150% recovery.</li> <li><strong>Proper dilution:</strong> Dilute samples appropriately to avoid inhibition or enhancement of the assay reaction. Validate dilution factors during assay development. Vortex lyophilized Endotoxin standards with vigor because endotoxin can adhere to surfaces. Conversely, LAL is an enzyme that should not be mixed viciously.</li> <li><strong>Release criteria:</strong> If you laboratory has a release criteria metric, it is advisable to include a sample at this concentration in your standard curve. This will ensure that you can have the highest level of confidence that unknown samples are above or below this threshold.</li> </ul> <h4 id="dataprocessing"><strong>Data Processing </strong></h4> <p>Pharmacopeia institutions require an initial linear regression to ensure that standards generate a curve with a r ≥ 0.98. After that, users can modify their curve fitting to best fit their standards. In general, a background-corrected, log-transformed, non-linear fit generates an accurate fit for end-point LAL assays with a dynamic range greater than 20x.</p> <h5><a href="https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Ffluorescence-based-bacterial-endotoxin-testing-poster.pdf" target="_blank" rel="noopener">Learn more about data processing for endotoxin assays>></a></h5> <p><strong> </strong></p> <h4 id="training"><strong>Training </strong></h4> <p>As with any assay, results are only as good as pipetting technique. Because assays are often time-sensitive, a positive displacement repeater pipette may minimize time and error. Assay workflows can often require multiple steps. Train to the specific assay you are working with – there can be multiple sources of contamination and error.</p> <h3 id="learnmore"><strong>Where can I learn more about emerging endotoxin methods? </strong></h3> <p>We’ve got you covered, whether you’re just starting out with endotoxin analysis or ready to dive into high-throughput applications and more.</p> <ul> <li><strong>Technology: </strong><a href="https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit.html?icid=fl-bid-qubit" target="_blank" rel="noopener">Qubit Fluorometric Quantification<br /> </a></li> <li><strong>Technology: </strong><a href="https://www.thermofisher.com/us/en/home/brands/product-brand/quant-it.html?icid=fl-bid-quantit" target="_blank" rel="noopener">Quant-iT Assays</a></li> <li><strong>Overview: </strong><a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/endotoxin-quantitation-removal.html" target="_blank" rel="noopener">Endotoxin Quantitation, Removal, and Detection</a></li> <li><strong>Overview: </strong><a href="https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/protein-purification/endotoxin-quantitation-removal/overview-of-endotoxin-testing-methods.html" target="_blank" rel="noopener">Endotoxin Testing Methods</a></li> <li><strong>Poster: </strong><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Fevaluation-of-endotoxin-contamination-testing-poster.pdf" target="_blank" rel="noopener">Evaluation of endotoxin contamination testing for LNP and AAV gene delivery systems</a></li> <li><strong>Poster: </strong><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Freliable-nucleic-acid-characterization-high-throughput-applications-poster.pdf" target="_blank" rel="noopener">A guide for reliable nucleic acid characterization in high-throughput applications</a></li> <li><strong>Poster: </strong><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Fendotoxin-detection-assay-validation-poster.pdf" target="_blank" rel="noopener">A validation study of Qubit and Quant-iT Endotoxin Detection Assays as an alternative method for bacterial endotoxin testing</a></li> <li><strong>Poster: </strong><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Ffluorescence-based-bacterial-endotoxin-testing-poster.pdf" target="_blank" rel="noopener">Fluorescence-based bacterial endotoxin testing – High sensitivity detection with a flexible and streamlined workflow</a></li> <li><strong>Poster: </strong><a href="https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FBID%2Fposters%2Fqubit-endotoxin-assay-poster.pdf" target="_blank" rel="noopener">Evaluation of a novel fluorescence-based approach to sensitive bacterial endotoxin testing</a></li> </ul> <p> </p> <p><em>Want to read more stories like this? </em><a href="https://www.thermofisher.com/us/en/home/global/forms/connect-science-subscribe.html?icid=bid_cbu_sbu_r01_co_cp1605_pjt10933_blg50592_0so_blg_il_awa_og_s00_C2SOncoBlogC2SSub" target="_blank" rel="noopener"><em>Subscribe to Connect to Science</em></a><em>, your portal for life science news.</em></p> <p> </p> <p>##</p> <p><em>For Research Use Only. 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