The study was supported by federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

1. Preparation of Nanoparticle Samples
<ol>
	<li>Prepare the study sample in LAL grade water.</li>
	<li>If the sample pH is outside of the 6-8 range, adjust the pH by using pyrogen-free sodium hydroxide or hydrochloric acid.</li>
	<li>Using LAL grade water prepare several dilutions of the study sample. Make sure that the highest dilution does not exceed maximum valid dilution (MVD). Refer to the discussion section for details about MVD estimation.</li>
</ol>
2. Preparation of Reagents Common Between LAL Formats
<ol>
	<li>Dilute concentrated sodium hydroxide stock using pyrogen-free LAL reagent water to prepare a working solution at a concentration of 0.1 N.</li>
	<li>Dilute concentrated hydrochloric acid stock using pyrogen-free LAL reagent water and prepare a working solution at a final concentration of 0.1 N.</li>
	<li>Preparation of the Control Standard Endotoxin (CSE)
	<ol>
		<li>Reconstitute the CSE according to the certificate of analysis supplied by the manufacturer. 
		NOTE: Refer to the discussion section for important notes regarding the information provided in the certificate. Refer to the Table of Materials for the details regarding catalog number and application of a given CSE formulation in different LAL formats.</li>
	</ol>
	</li>
	<li>Preparation of the LAL Reagent
	<ol>
		<li>Reconstitute the LAL reagent according to the certificate of analysis provided by the manufacturer. 
		NOTE: Refer to the discussion section for important details regarding LAL reagent preparation. Refer to the Table of Materials for the details regarding catalog number and application of a given LAL reagent formulation in different LAL format.</li>
	</ol>
	</li>
</ol>
3. Turbidity LAL Assay
<ol>
	<li>Preparation of the Calibration Standards
	<ol>
		<li>Using 900 &#181;L of LAL grade water and 100 &#181;L of CSE, prepare as many intermediate dilutions as needed to enable the preparation of a calibration standard with a concentration range from 0.001 to 1 EU/mL.</li>
		<li>First label tubes and add 900 &#956;L of LAL-grade water into each tube. Then add 100&#160;&#956;L of 10 EU/mL solutionn to prepare calibration standard with concentration of 1EU/mL.</li>
		<li>Repeat the serial 10-fold dilution as described above to prepare three lower calibration standards. Verify that four calibration standards ranging from 0.001 to 1 EU/mL have been prepared.</li>
	</ol>
	</li>
	<li>Preparation of the Quality Controls
	<ol>
		<li>Prepare a 0.05 EU/mL quality control by combining 50 &#181;L of the 1 EU/mL CSE solution with 950 &#181;L of LAL-grade water. 
		NOTE: Refer to the discussion section for details regarding control preparation.</li>
	</ol>
	</li>
	<li>Preparation of Inhibition/Enhancement (IEC) Controls
	<ol>
		<li>Prepare IEC with concentration of 0.05 EU/mL by combining 25 &#181;L of the 1 EU/mL CSE solution and 475 &#181;L of the test nanomaterial at a given dilution. 
		NOTE: Refer to the discussion section for additional details.</li>
	</ol>
	</li>
	<li>Experimental procedure
	<ol>
		<li>Allow the instrument to warm up by turning it on approximately 30 min in advance. Set-up the detection wavelength to 660 nm as this is appropriate for the turbidity LAL.</li>
		<li>Sign in by typing the username and password.</li>
		<li>Open the software (Table of Materials) by clicking on the corresponding icon on a computer screen.</li>
		<li>Select Collect data on the software home screen. Enter the test ID and data group information into the corresponding space in the General tab on the home screen.</li>
		<li>Click the Hardware tab. Choose the instrument type from a dropdown menu.</li>
		<li>Set-up the detection wavelength to 660 nm as this is appropriate for the turbidity LAL by chosing the LAL turbidity method.</li>
		<li>Verify that a serial number, system ID and serial port information appear on the screen. Click OK. Click OK one more time to confirm.</li>
		<li>Enter the sample ID in the same order the sample is tested. Use default buttons to enter the negative control, standard curve and test samples.&#160;</li>
		<li>Prepare duplicate tubes for each sample and add 200 &#181;L (test ratio&#160;4:1) or 100 &#181;L (test ratio 1:1) of negative control (water), calibration standards, quality control, IEC and test nanoparticles into pre-labeled glass tubes.</li>
		<li>Add 50 &#181;L (test ratio 4:1) or 100 &#181;L (test ratio 1:1) of LAL reagent to first test vial, vortex it briefly, and insert into test slot in the instrument carousel. If the 1:1 ratio is used, the volume of the LAL reagent is 100 &#181;L.</li>
		<li>Repeat the procedure described above for other samples. Process samples one at a time. 
		NOTE: Refer to the discussion section for more details.</li>
	</ol>
	</li>
</ol>
4. Chromogenic LAL
<ol>
	<li>Preparation of Calibration Standards
	<ol>
		<li>Using 900 &#181;L of LAL grade water and 100 &#181;L of CSE, prepare as many intermediate dilutions as needed to enable the preparation of a calibration standard with a concentration of 1 EU/mL.</li>
		<li>Using 900 &#181;L LAL-grade water and 100 &#181;L of the 1 EU/mL calibration standard, prepare a second calibration standard at a concentration of 0.1 EU/mL.</li>
		<li>Repeat the serial 10-fold dilution as described above to prepare two lower calibration standards. Verify that four calibration standards ranging from 0.001 to 1 EU/mL have been prepared.</li>
	</ol>
	</li>
	<li>Preparation of Quality Controls.
	<ol>
		<li>Prepare a 0.05 EU/mL quality control by combining 50 &#181;L of the 1 EU/mL CSE solution with 950 &#181;L of LAL-grade water. 
		NOTE: Refer to the discussion section for details regarding control preparation.</li>
	</ol>
	</li>
	<li>Preparation of Inhibition/Enhancement (IEC) Controls
	<ol>
		<li>Prepare 0.05 EU/mL by combining 25 &#956;L of the 1 EU/mL CSE solution with 475 &#956;L of test nanomaterial. 
		NOTE: Refer to the discussion section for additional details.</li>
	</ol>
	</li>
	<li>Experimental procedure
	<ol>
		<li>Allow the instrument to warm up by turning it on approximately 30 min in advance. Set-up the detection wavelength to 405 nm as this is appropriate for the turbidity LAL.</li>
		<li>Open the software by clicking on the corresponding icon on a computer screen. Sign in by typing the username and password.</li>
		<li>Select Collect data on the software home screen. Enter test ID and data group information into corresponding space in the General tab on the home screen.</li>
		<li>Click the Hardware tab. Choose the instrument type from a dropdown menu. Choose the instrument.</li>
		<li>Verify that a serial number, system ID and serial port information appear on the screen. Click OK. Click OK one more time to confirm.</li>
		<li>Enter the sample ID in the same order the sample is tested. Use default buttons to enter negative control, standard curve and test samples.</li>
		<li>Prepare duplicate tubes for each sample and add 200 &#181;L (test ratio&#160;4:1) or 100 &#181;L (test ratio 1:1) of negative control (water), calibration standards, quality control, IEC and test nanoparticles into pre-labeled glass tubes.</li>
		<li>Add 50 &#181;L (test ratio 4:1) or 100 &#181;L (test ratio 1:1) of LAL reagent to first test vial, vortex it briefly, and insert into test slot in the instrument carousel. If the 1:1 ratio is used, the volume of the LAL reagent is 100 &#181;L.</li>
		<li>Repeat the procedure described above for other samples. Process samples one at a time.</li>
	</ol>
	</li>
</ol>
5. Gel-Clot LAL
NOTE: This assay identifies the presence of endotoxins in the sample based on the visual observation and detection of a clot in the reaction tube. The experimental steps are described below. Use a bench sheet to record the results. This bench sheet is not mandatory, and other ways of recording the assay results are also acceptable. An example of such a bench sheet is provided in supplementary materials for the convenience of a reader. Lambda (l) is the sensitivity of the gel-clot assay and is 0.03 EU/mL.
<ol>
	<li>Label as many reaction tubes as needed to accommodate the number of analyzed test samples. Refer to the bench sheet for details about the number of replicates used in step 1, step 2 and step 3 of the assay.</li>
	<li>Aliquot 100 &#956;L of water, controls or test sample per tube.</li>
	<li>Prepare CSE such that the final concentration is equal to 4&#955;.</li>
	<li>Combine 100 &#956;L of the standard mentioned above with 100 &#956;L of water or test sample to achieve the final concentration of CSE of 2&#955;. Repeat three more times to achieve lambda and half-lambda and a quarter lambda.</li>
	<li>Make sure that the temperature in the water bath is 37 &#176;C.</li>
	<li>Add 100 &#956;L of lysate per test tube, vortex briefly and place the rack with all the tubes into the water bath for 1 h.</li>
	<li>Invert the tube with a smooth motion.</li>
	<li>Manually record results using &#8220;+&#8221; (firm clot) or &#8220;-&#8220; (no clot or loose clot) on the bench sheet.</li>
	<li>Proceed with the analysis according to the USP BET 854;use the bench sheet as supporting material</li>
</ol>

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The authors have nothing to disclose.

The information provided in this protocol has been described before15,26 and relies on several regulatory documents published by the US Food and Drug Administration (US FDA or FDA) and United States Pharmacopoeia (USP)4,5,6,27, and is also available on the NCL website20 in protocols STE1.2 (turbidity LAL), STE1.3 (gel-clot LAL) and STE1.4 (chromogenic LAL).
Test-nanomaterials are prepared either in LAL reagent water or sterile, pyrogen-free PBS. The pH of the study sample is important because low (&#60; 6) and high (&#62; 8) pH will interfere with the optimal performance of the lysate. If the pH of the test-nanoparticle is outside of the 6-8 range, it can be adjusted using pyrogen-free sodium hydroxide or hydrochloric acid. When such adjustment is performed, it is vital to avoid sample contamination from microelectrode. Therefore, to perform this procedure, a small aliquot of the sample is removed into a separate tube and used to measure the pH.
When the sample is prepared in PBS or another buffer, the blank buffer is also included in this test. The concentration of each nanomaterial is case-specific. The test is used to estimate the amount of contaminating endotoxin per milligram of the active pharmaceutical ingredient (API). However, depending on the type of nano-formulation, the concentration can also be estimated in mg of the total formulation or total element (e.g., gold, silver or iron for gold, silver, and iron oxide nanoparticles, respectively). The sample is tested from the stock using several dilutions not exceeding the MVD. All dilutions of the test-nanoparticles are prepared using LAL-grade water.
Three parameters are used to calculate the MVD. They are the endotoxin limit (EL), the sample concentration, and the assay sensitivity (&#955;)4. Use the following formula to calculate endotoxin limit (EL): EL=K/M, where K is a threshold pyrogen dose (e.g., 5 EU/kg as discussed in the introduction) and M is the maximum dose of the test nanomaterial intended for administration per kilogram of body weight in a single hour4. As discussed above, the estimation of EL for nanomaterials used as radiopharmaceutical or as medical device relies on a threshold pyrogenic dose or formula, which is different from 5/M. These details are mentioned in the introduction and could be further reviewed in the USP and US FDA guidelines4,5,6. The recommended endotoxin limit for ophthalmic solutions and intraocular devices is provided in the US FDA guideline for these products6. When an animal model (e.g., mouse) was used to establish the dose of nanoformulation, this information is used to estimate the so-called human equivalent dose (HED). The HED is calculated by dividing the animal dose by a conversion factor, which is specific to each animal species. For example, the conversion factor is 12.3, 6.2 and 3.1 for the mouse, the rat, and the rabbit, respectively27. The conversion procedure and rationale for using it are described in detail in the US FDA guideline27. Cancer therapeutics are often dosed per surface body area expressed in mg/m2. One can either follow the USP guideline to calculate EL for drugs dosed in milligram per square meter or convert this dose to mg/kg range. The dose in mg/m2 can be adjusted to the dose in mg/kg using a species-specific conversion factor27. For an adult human, it is 37 and indicated as Km to refer to the mass constant27. The km factor has units of kg/m2; it is equal to the body weight in kilograms (kg) divided by the surface area in square meters (m2)27. For example, a human dose or HED of 74 mg/m2 corresponds to 2 mg/kg or 74/37. To determine MVD, use the following formula, which is also available from the USP BET 85 standard: MVD= (EL x sample concentration)/&#955;)4. In a hypothetical scenario, a nanoparticle sample concentration is 10 mg/mL and its maximum dose in the mouse is 615 mg/kg. In this case the HED is 615/12.3=50 mg/kg; EL for all routes except intrathecal is 0.1 EU/mg (5 EU/kg/50 mg/kg) and MVD is 1,000 ((0.1 EU/mg x 10 mg/mL)/0.001 EU/mL).
Often, when one needs to evaluate endotoxin contamination in a research grade nanomaterial, the dose information is not available. There is no harmonized procedure for how to estimate the MVD and EL for these materials. This case study performs the test directly from stock (commonly the concentration of this solution is 1 mg/mL) and at several dilutions, usually 5-, 50-, 500- and 5,000- folds. When the dose, the route of administration, the sample concentration and the infusion time for the given test nanomaterial change, the EL and MVD also change. Therefore, one has to evaluate the information and perform estimation of EL and MVD in the context of other parameters related to the amount, time, intended route of administration and concentration of the given formulation.
It is important to note that the catalog number and the product specifications (e.g., potency, amounts per vial) of the CSE are different for different LAL formats. The CSE used in turbidity LAL can also be used in a gel-clot LAL. However, the CSE for a chromogenic LAL is specific to this assay format. The instructions provided below are general and applicable to the CSE used in all assay formats. The CSE is an E. coli lipopolysaccharide (LPS) supplied as a lyophilized powder. This standard is certified by the manufacturer against a Reference Standard Endotoxin (RSE) with a known potency. The contents of the vial containing CSE should be reconstituted with ~3.2 - 5.0 mL of pyrogen-free LAL reagent water. In addition to the difference between CSE used for different assay formats, the volume of reconstitution is also specific to each lot of this standard. Therefore, one has to calculate the final concentration of the stock CSE solution for each lot of the standard. The calculation is performed based on the potency of the standard and the amount contained in each vial. The certificate of analysis specific for each lot of endotoxin standard contains the information about the potency and amount per vial. One has to rigorously vortex the standard for 30-60 seconds, both during the reconstitution and during the use. It is also recommended to perform the reconstitution using 5-10 min settling times and over a 30-60 min time frame to ensure that all lyophilized material goes into solution. Before using in the assay, equilibrate the stock CSE to room temperature. After the reconstitution, the CSE stock can be refrigerated for storage and is stable for four weeks.
Similar to the CSE, the LAL reagent is also specific for each format. The instructions provided below are general and applicable to all LAL assay formats. The LAL reagent is provided as a lyophilized powder. The manufacturer&#39;s recommendations are followed to reconstitute each vial. Either LAL grade water or buffer inhibiting beta-glucans can be used. The buffer inhibiting beta-glucans is preferred in this case-study because it allows excluding the interference from beta-glucans. This false-positive interference is very common in nanomaterials because cellulose-acetate filters are commonly used during nanomaterial synthesis and serve as a source of glucan contamination15. Most vials will require reconstitution to a final volume of 5 mL. Based on over 10 years of the authors&#39; experience with this assay, it has been noticed that the inclusion of this buffer slows down the reaction and may require increasing the maximum onset time in the instrument settings to allow the lowest calibrator at a concentration of 0.001 EU/mL to develop.
To ensure accurate dilution during the preparation of standards, it is recommended to use 10-fold dilution steps. Serial 10-fold dilutions can be prepared by spiking 100 &#181;L of the stock or a standard with higher concentration into 900 &#181;L of pyrogen-free water. Since the concentration of the CSE stock is usually high (~1,000 EU/mL), preparation of intermediate dilutions with concentrations 100 and 10 EU/mL is recommended before preparing the assay calibrator with a concentration of 1 EU/mL. Two ratios between the test sample and lysate are described in this protocol. Even though both ratios are commonly used, the curve linearity is acceptable but not ideal when the 4:1 ratio and the buffer inhibiting beta-glucans (see the Table of Materials) are used.
The same rules as described above regarding the calibration standard preparation also apply to the preparation of quality controls (QC). These controls are used to verify that the assay is functioning correctly. It is prepared by spiking the known amount of CSE into the pyrogen-free water. The concentration of QC is usually chosen in the middle of the assay range. The range of the turbidity LAL assay is 0.001 to 1 EU/mL. Therefore, a QC at a concentration of 0.05 EU/mL is used. Other concentrations within the assay range can also be used in the preparation of the quality control.
The inhibition enhancement control (IEC) is also known as positive product control (PPC). It is prepared by spiking a known concentration of CSE into the test sample. The concentration of IEC (PPC) is usually chosen in the middle of the assay range. The range of the turbidity LAL assay is 0.001 to 1 EU/mL. Therefore, the IEC at a concentration of 0.05 EU/mL is used. To prepare this IEC one needs to combine 50 &#181;L of the 1 EU/mL CSE solution with 950 &#181;L of test-nanoparticle. The IEC is prepared for each dilution of a test-nanomaterial. For example, if a nano-formulation is tested at three dilutions (1:50, 1:500 and 1:5,000), one has to prepare three IECs, one for each of these dilutions. Other concentrations within the assay range can also be used to prepare the IEC sample. This control is used to understand the validity of the test results for the given nano-formulation. According to the USP, the test-results are valid if the spike recovery of IEC (PPC) is between 50 and 200%4. Spike recovery less than 50% means that the test-nanomaterial inhibits endotoxin detection and, therefore, the test result underestimates endotoxin contamination and is invalid. Spike recovery over 200% suggests that the test-material enhances the assay result or contains a too high level of the contaminating endotoxin. In the case of enhancement, the assay result is also inaccurate. Examples of such interferences and some ways for overcoming them have been described earlier12,15.
When performing turbidity and chromogenic assays, it is recommended to use a repeater pipette to add the LAL reagent to all samples. The average instrument operation time is 7200 seconds. However, the reaction can be faster or slower with certain lots of the lysate. In cases, when the reaction is slow, one may need to change the instrument settings to 9600s or longer. This change will allow the lowest calibrator to develop.
PEGylated liposomal doxorubicin is a formulation containing an active pharmaceutical ingredient (API) at a concentration of 2 mg/mL. It is used in the clinic as a dose of 50 mg/m2. To convert this dose to mg/kg range, it is divided by factor 3727. The dose of PEGylated liposomal doxorubicin in mg/kg, therefore, is 1.35. To calculate EL, 5 (the threshold pyrogen dose in EU/kg) is divided by 1.35 (the dose of Doxil in mg/kg) and obtain 3.7 EU/mg4. This number means that PEGylated liposomal doxorubicin at the dose of 1.35 mg/kg can be safely administered per single hour if endotoxin in the formulation does not exceed 3.7 EU/mg, where mg refers to the API.
Next, use this information to calculate the MVD for LAL assays. The sensitivity (lambda) of the turbidity, chromogenic and gel-clot assays presented in this study is 0.001, 0.001 and 0.03 EU/mL, respectively. Therefore, MVD is 7,407 for turbidity and chromogenic assays ((5 EU/kg x 2 mg/mL)/0.001 EU/mL), and 247 for the gel-clot assay ((5 EU/kg x 2 mg/mL)/0.03 EU/mL).
Doxorubicin has a broad absorbance spectrum that overlaps with assay wavelength of the chromogenic LAL28,29. Moreover, most liposome formulations are turbid and appear milky. This inherent turbidity is the very common reason for the interference of liposomes with turbidity assay. In the case of PEGylated liposomal doxorubicin, the interference due to the turbidity was overcome by dilution five as evidenced by the spike recovery values between 50 and 200% (Table 1). However, at the same dilution doxorubicin concentration was still high enough to interfere with the chromogenic LAL (Table 1). Two subsequent dilutions (50 and 500) helped to overcome PEGylated liposomal doxorubicin interference with chromogenic LAL. Since the gel-clot assay is independent of turbidity and sample color, and the level of endotoxin in the formulation is within the gel-clot range, the PEGylated liposomal doxorubicin did not interfere with the gel-clot assay at all tested dilutions. In this case study, several dilutions of PEGylated liposomal doxorubicin were used for these assays. The dilutions 5, 50 and 500 for turbidity and chromogenic as well as 50, 100 and 200 for the gel-clot assay were chosen because they are within the MVD. The MVD of the gel-clot assay is lower than that of the turbidity and chromogenic LALs because the sensitivity of this assay is also lower. If the formulation interfered with the LAL at dilution 500 in turbidity and chromogenic assays, the analysis at higher dilutions (e.g., 5,000, 6,000, 7,000 or 7,407) could be performed. Since the level of endotoxin obtained at dilution 50 in the gel-clot assay was below the EL, the analysis of this formulation at lower dilutions was not performed. However, in other cases, the testing could be continued at Dilutions 20, 10, 5 or 2 to identify the lowest not interfering dilution and understand whether the level of endotoxin in the formulation is below the EL.
It is important to mention that for the gel-clot assay it is essential to turn off the water circulation function in the water bath for the duration of this assay or use a water bath without such option because water flow damages the clot and may affect the accuracy of the gel-clot LAL assay. It is not always possible to overcome nanoparticle interference with LAL assays by increasing the sample dilutions. Strategies and some approaches for overcoming various types of interference and examples of nanoparticles commonly representing a problem for LAL assays have been discussed by other groups and us elsewhere12,13,14,15,30,31. The analysis presented in this manuscript is based on a model formulation. The experience with other nanoformulations may be different.

An endotoxin is a building block of the Gram-negative bacterial cell wall1,2. It can activate the immune cells at very low (picogram) concentrations1,2. The proinflammatory mediators (cytokines, leukotrienes, eicosanoids, etc.) produced by the cells in response to an endotoxin are responsible for fever, hypotension, hypertension, and more severe health problems including multiple organ failure1,2,3. The severity of the immune-mediated side-effects triggered by the endotoxin depends on its potency determined by the endotoxin composition and structure and measured in international endotoxin units (IUs or EUs)3. The number of these units per kilogram of body weight is used to set a threshold pyrogenic dose of endotoxin. This dose is 5 EU/kg for drug products administered via all routes but the intrathecal route. Drugs dosed per square meter of body surface, intraocular fluids, radiopharmaceuticals, and products administered via intrathecal route have a different threshold pyrogenic dose, which is 100 EU/m2, 0.2 EU/mL, 175 EU/V (where V is the volume of the product intended for administration), and 0.2 EU/kg, respectively4. More details about the threshold pyrogenic dose for various drug products and devices are provided and discussed elsewhere4,5,6.
Animals vary widely in their sensitivity to endotoxin-mediated reactions. Humans, non-human primates, and rabbits are among the species most extremely sensitive to endotoxins3. To avoid endotoxin-mediated side effects in patients and prevent inaccurate conclusions of preclinical toxicity and efficacy studies, it is essential to accurately detect and quantify endotoxins in both clinical and pre-clinical grade formulations. Several currently available methods can achieve this task. One of them is the Limulus Amoebocyte Lysate (LAL) assay, which is commonly used worldwide to screen biomedical products for the potential endotoxin contamination as well as to detect bacterial infections7,8,9. The lysate is prepared from amoebocytes, the cells present in the blood of horseshoe crabs Limulus polyphemus residing in the east shore of the continent of North America7. Interestingly, there are a few different species of horseshoe crabs (Tachypleus gigas and Tachypleus tridentatus) in Asia10. The Tachypleus Amoebocyte Lysate (TAL) is used in several Asian countries for the detection of endotoxin similar to how the LAL is used in other cuntries10. The lysates (LAL and TAL) contain a group of proteins that upon activation confer protease activity. One of these proteins, the so-called Factor C is activated upon contact with endotoxin. Activated Factor C cleaves Factor B, which in turn also becomes a protease and cleaves a pro-clotting enzyme to produce a clotting enzyme. The result of this chain of reactions is the formation of a gel, an increase in the sample turbidity and, in the presence of a chromogenic substrate, the appearance of a colored product, which serve as a foundation for gel-clot, turbidity, and chromogenic assays, respectively. While there is no mandatory LAL format, the US Food and Drug Administration (FDA) explains in the guidance for industry document, that in case of discrepancy in the test results between different LAL formats, the decision is made based on the gel-clot assay5.
Many commonly used laboratory chemicals (e.g., EDTA) and known drug products (e.g., penicillin) interfere with LAL assays11. The interference is usually identified by assessing the recovery of the endotoxin standard spiked at a known concentration into a solution containing the test material. If the spike recovery is less than 50% or more than 200%, then the result of the LAL assay for the given test material is invalid due to the inhibition or enhancement, respectively4. Nanotechnology-based formulations are often complex and interfere with the LAL through a variety of mechanisms12,13,14. Many approaches have been described to overcome the interference: sample reconstitution in specific buffers and surfactants, protein inactivation by heating, destruction of lipid-based hollow materials by heating and supplementing the sample with excess divalent cations5,12,13,14,15. Alternative methods for situations when LAL interference cannot be overcome have also been described: ELISA, a HEK-TLR4 reporter cell line assay, and mass spectrometry16,17,18,19.
Herein, experimental procedures for conducting gel-clot, turbidity, and chromogenic LAL assays are described. These assays are also available on the Nanotechnology Characterization Lab (NCL) website20 in protocols STE1.2 (turbidity LAL), STE1.3 (gel-clot LAL) and STE1.4 (chromogenic LAL). It is recommended to conduct at least two different formats to characterize the same nano-formulation. When results of the turbidity and chromogenic LAL disagree, the gel-clot results are considered5. When results of two LAL formats disagree, additional studies using either monocyte activation test (MAT) or rabbit pyrogen test (RPT) to verify LAL findings are conducted21. It is important to note that each method used for endotoxin detection and pyrogenicity assessment has advantages and limitations21,22,23,24. Recognizing limitations of the procedure used to characterize a given nanotechnology formulation is essential to obtain scientific justification for the use of the procedure optimal for that nano-formulation.
In this study, PEGylated liposomal doxorubicin was used as a model nanoparticle formulation. This formulation has been approved by the US FDA in 1995 and used for treating cancer patients worldwide25.

<table><tbody><tr><td>&lt;strong&gt;Turbidity LAL Assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S2770</td><td>When needed, it is used to adjust sample pH to be between 6-8</td></tr><tr><td>Hydrochloric acid</td><td>Sigma</td><td>H9892</td><td>When needed, it is used to adjust sample pH to be between 6-8</td></tr><tr><td>LAL Reagent</td><td>Associates of Cape Cod</td><td>T0051</td><td>This reagent can be used with turbidity assay only</td></tr><tr><td>Control Endotoxin Standard</td><td>Associates of Cape Cod</td><td>E0005</td><td>This reagent can be used with turbidity and gel-clot assays</td></tr><tr><td>LAL grade water</td><td>Associates of Cape Cod</td><td>WP0501</td><td>This reagent can be used with any LAL format</td></tr><tr><td>Glucashield Buffer</td><td>Associates of Cape Cod</td><td>GB051-25</td><td>Used to prevent false-positive response from beta-glucans</td></tr><tr><td>Disposable endotoxin-free glass dilution tubes 12 x 75 mm</td><td>Associates of Cape Cod</td><td>TB240</td><td>These tubes can be used with all three assays</td></tr><tr><td>Disposable endotoxin-free glass reaction tubes 8 x 75 mm</td><td>Associates of Cape Cod</td><td>TK100</td><td>These tubes can be used with turbidity and chromogenic assays</td></tr><tr><td>Pyrogen-free tips with volumes 0.25 and 1.0 mL</td><td>RAININ</td><td>PPT25, PPT10</td><td>Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed</td></tr><tr><td>Pyrogen-free microcentrifuge tubes, 2.0 mL</td><td>Eppendorf</td><td>22600044</td><td>Other equivalent supplies can be used</td></tr><tr><td>Pyrogen-fee combitips, 5mL</td><td>Eppendorf</td><td>30089669</td><td>Other equivalent supplies can be used</td></tr><tr><td>Repeat pipettor</td><td>Eppendorf</td><td>4982000020</td><td>Other equivalent supplies can be used</td></tr><tr><td>Microcetrifuge</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Refrigerator, 2-8 C</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Vortex</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Freezer, -20 C</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Pyros Kinetix or Pyros Kinetix Flex reader</td><td>Associates of Cape Cod</td><td>PKF96</td><td>Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions</td></tr><tr><td>&lt;strong&gt;Chromogenic LAL Assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pyrochrome LAL Reagent</td><td>Associates of Cape Cod</td><td>CG1500-5</td><td>This reagent is specific to the Chromogenic Assay</td></tr><tr><td>Control Endotoxin Standard</td><td>Associates of Cape Cod</td><td>EC010</td><td>This standard is different than that used for turbidity and gel-clot LALs; it is optimized for optimal performance in the chromogenic assay</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S2770</td><td>When needed, it is used to adjust sample pH to be between 6-8</td></tr><tr><td>Hydrochloric acid</td><td>Sigma</td><td>H9892</td><td>When needed, it is used to adjust sample pH to be between 6-8</td></tr><tr><td>LAL grade water</td><td>Associates of Cape Cod</td><td>WP0501</td><td>This reagent can be used with any LAL format</td></tr><tr><td>Glucashield Buffer</td><td>Associates of Cape Cod</td><td>GB051-25</td><td>Used to prevent false-positive response from beta-glucans</td></tr><tr><td>Disposable endotoxin-free glass dilution tubes 12 x 75 mm</td><td>Associates of Cape Cod</td><td>TB240</td><td>These tubes can be used with all three assays</td></tr><tr><td>Disposable endotoxin-free glass reaction tubes 8 x 75 mm</td><td>Associates of Cape Cod</td><td>TK100</td><td>These tubes can be used with turbidity and chromogenic assays</td></tr><tr><td>Pyrogen-free tips with volumes 0.25 and 1.0 ml</td><td>RAININ</td><td>PPT25, PPT10</td><td>Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed</td></tr><tr><td>Pyrogen-free microcentrifuge tubes, 2.0 mL</td><td>Eppendorf</td><td>22600044</td><td>Other equivalent supplies can be used</td></tr><tr><td>Pyrogen-fee combitips, 5mL</td><td>Eppendorf</td><td>30089669</td><td>Other equivalent supplies can be used</td></tr><tr><td>Repeat pipettor</td><td>Eppendorf</td><td>4982000020</td><td>Other equivalent supplies can be used</td></tr><tr><td>Microcetrifuge</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Refrigerator, 2-8 C</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Vortex</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Freezer, -20 C</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Pyros Kinetix or Pyros Kinetix Flex reader</td><td>Associates of Cape Cod</td><td>PKF96</td><td>Other instruments can be used. However, LAL reagents and endotoxin standards used in this assay may require optimization. When other instrumentation is used, please refer to the instrument and LAL kit manufacturers for instructions</td></tr><tr><td>&lt;strong&gt;Gel-Clot LAL Assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>LAL Reagent</td><td>Associates of Cape Cod</td><td>G5003</td><td>This reagent is specific to the gel-clot assay</td></tr><tr><td>Control Endotoxin Standard</td><td>Associates of Cape Cod</td><td>E0005</td><td>This reagent can be used with turbidity and gel-clot assays</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma</td><td>S2770</td><td>When needed, it is used to adjust sample pH to be between 6-8</td></tr><tr><td>Hydrochloric acid</td><td>Sigma</td><td>H9892</td><td>When needed, it is used to adjust sample pH to be between 6-8</td></tr><tr><td>LAL grade water</td><td>Associates of Cape Cod</td><td>WP0501</td><td>This reagent can be used with any LAL format</td></tr><tr><td>Glucashield Buffer</td><td>Associates of Cape Cod</td><td>GB051-25</td><td>Used to prevent false-positive response from beta-glucans</td></tr><tr><td>Disposable endotoxin-free glass dilution tubes 12 x 75 mm</td><td>Associates of Cape Cod</td><td>TB240</td><td>These tubes can be used with all three assays</td></tr><tr><td>Disposable endotoxin-free glass reaction tubes 10 x 75 mm</td><td>Associates of Cape Cod</td><td>TS050</td><td>These tubes are for use with the gel-clot assay</td></tr><tr><td>Pyrogen-free tips with volumes 0.25 and 1 mL</td><td>RAININ</td><td>PPT25, PPT10</td><td>Tips and pipettes may adsorb endotoxin and release leachables which interfere with LAL assay. These RAININ tips are used because their optimal performance in the LAL assay was verified and confirmed</td></tr><tr><td>Pyrogen-free microcentrifuge tubes, 2.0 mL</td><td>Eppendorf</td><td>22600044</td><td>Other equivalent supplies can be used</td></tr><tr><td>Pyrogen-fee combitips, 5mL</td><td>Eppendorf</td><td>30089669</td><td>Other equivalent supplies can be used</td></tr><tr><td>Repeat pipettor</td><td>Eppendorf</td><td>4982000020</td><td>Other equivalent supplies can be used</td></tr><tr><td>Microcetrifuge</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Refrigerator, 2-8 C</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Vortex</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Freezer, -20 C</td><td>any brand</td><td></td><td>Any brand can be used</td></tr><tr><td>Water bath, 37 C</td><td>any brand</td><td></td><td>Any brand can be used, however, it is important either to switch off water circulation or use non-circualting water bath because water flow will affect clot formation and lead to false-negative results</td></tr></tbody></table>

detection of endotoxin in nano-formulations using limulus amoebocyte lysate (lal) assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

The example of data generated after testing this formulation in LAL assays is shown in Table 1. PEGylated liposomal doxorubicin interfered with chromogenic LAL at dilution 5. However, this interference was overcome by greater dilutions. Spike recovery was between 50 and 200% when this formulation was tested at dilutions 50 and 500 in turbidity and chromogenic LAL, as well as at dilution 5 in turbidity LAL. When adjusted by the dilution factor, the results were consistent between dilutions in both assays. Moreover, the results were consistent between the three assay formats.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Test sample</td>
			<td>Turbidity LAL, EU/mg (spike recovery,%)</td>
			<td>Chromogenic LAL, EU/mg, (spike recovery,%)</td>
			<td>Gel-clot LAL, EU/mg (test valid, Yes or No)</td>
		</tr>
		<tr>
			<td>PEGylated liposomal doxorubicin (see the Table of Materials)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dilution 5</td>
			<td>0.01 (141)</td>
			<td>interference</td>
			<td>&#60; 0.75 (Y)</td>
		</tr>
		<tr>
			<td>Dilution 50</td>
			<td>&#60; 0.025 (187)</td>
			<td>0.029 (82)</td>
			<td>&#60; 1.5 (Y)*</td>
		</tr>
		<tr>
			<td>Dilution 500</td>
			<td>&#60; 0.25 (182)</td>
			<td>&#60; 0.25 (86)</td>
			<td>&#60; 3 (Y)**</td>
		</tr>
	</tbody>
</table>
Table 1:&#160;Detection of endotoxin in PEGylated liposomal doxorubicin using LAL assays. PEGylated liposomal doxorubicin was tested using turbidity, chromogenic and gel-clot LAL assays. Sample interference was estimated using IECs. Spike recovery is shown in parenthesis. Values between 50 and 200% are considered acceptable according to the USP BET 85 standard for endotoxin detection4. * and ** the test results of this sample were obtained at dilutions 100 and 200, respectively. The gel-clot assay dilutions are different from those used in chromogenic and turbidity LAL because sensitivity and maximum valid dilution of this assay are lower.

Watch this Scientific Journal Video about Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays at JoVE.com

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

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JoVE Journal

Bioengineering

14.5K Views.  Frederick National Laboratory for Cancer Research sponsored by National Cancer Institute. Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Video: Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Environment

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development and implementation of biomedical devices and tissue engineering constructs. Macrophages, an innate immune cell, are key players in the foreign body reaction because they remain at the implant site for the lifetime of the device, and are commonly studied to gain an understanding of this detrimental host response. Many biomaterials researchers have shown that adsorbed protein layers on implanted materials influence macrophage behavior, and subsequently impact the host response. The methods in this paper describe an in vitro model using adsorbed protein layers containing cellular damage molecules on polymer biomaterial surfaces to assess macrophage responses. An NF-&#1082;B/AP-1 reporter macrophage cell line and the associated colorimetric alkaline phosphatase assay were used as a rapid method to indirectly examine NF-&#1082;B/AP-1 transcription factor activity in response to complex adsorbed protein layers containing blood proteins and damage-associated molecular patterns, as a model of the complex adsorbed protein layers formed on biomaterial surfaces in vivo.

This protocol provides researchers with a rapid, indirect method of measuring TLR-dependent NF-&#1082;B/AP-1 transcription factor activity in a murine macrophage cell line in response to a variety of polymeric surfaces and adsorbed protein layers that model the biomaterial implant microenvironment.

The persistent inflammatory host response to an implanted biomaterial, known as the foreign body reaction, is a significant challenge in the development ...

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis and septic shock. The early identification of an infectious process that is rapidly evolving to a critical illness might prompt a quicker and more intensive treatment, thereby potentially leading to better patient outcomes. The Endotoxin Activity (EA) assay can be used at the bedside as a reliable biomarker of systemic endotoxemia. The detection of elevated endotoxin activity levels has been repeatedly shown to be associated with an increased disease severity in patients with sepsis and septic shock. The assay is quick and easy to perform. Briefly, after sampling, an aliquot of whole blood is mixed with an anti-endotoxin antibody and with added LPS. Endotoxin activity is measured as the relative oxidative burst of primed neutrophils as detected by chemioluminescence. The assay&#39;s output is expressed on a scale from 0 (absent) to 1 (maximal) and categorized as &#8220;low&#8221; (&#60;0.4 units), &#8220;intermediate&#8221; (0.4&#8211;0.59 units), or &#8220;high&#8221; (&#8805;0.6 units). The detailed methodology and rationale for the implementation of the EA assay are reported in this manuscript.

We hereby present a protocol to measure at the bedside the endotoxin activity of human whole blood samples. The Endotoxin Activity assay is a simple test to perform and may be a useful biomarker in critically ill patients with sepsis.

Lipopolysaccharide, also known as endotoxin, is a fundamental component of gram-negative bacteria and plays a crucial role in the development of sepsis ...

Medicine

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and ...

Immunology and Infection

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce validly immune responses. However, establishing a series of comprehensive protocols to evaluate the immune response of a novel nanoemulsion adjuvant vaccine is vital. Therefore, this article features a rigorous procedure to determine the physicochemical characteristics of a vaccine (by transmission electron microscopy [TEM], atomic force microscopy [AFM], and dynamic light scattering [DLS]), the stability of the vaccine antigen and system (by a high-speed centrifuge test, a thermodynamic stability test, SDS-PAGE, and western blot), and the specific immune response (IgG1, IgG2a, and IgG2b). Using this approach, researchers can evaluate accurately&#160;the protective effect of a novel nanoemulsion adjuvant vaccine in a lethal MRSA252 mouse model. With these protocols, the most promising nanoemulsion vaccine adjuvant in terms of effective adjuvant potential can be identified. In addition, the methods can help optimize novel vaccines for future development.

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus MRSA Infection

The present protocol prepares and evaluates the physical properties, immune response, and in vivo protective effect of a novel nanoemulsion adjuvant vaccine.

Nanoemulsion adjuvant vaccines have attracted extensive attention because of their small particle size, high thermal stability, and ability to induce ...

Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

Source: Neun, B. W. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58830">Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays.</a> J. Vis. Exp. (2019)
This video describes the turbidimetric format of the Limulus amoebocyte lysate, or LAL, assay to estimate endotoxin contamination in PEGylated liposomal doxorubicin nano-formulations. The turbidity of the solution following the reaction of the endotoxin with the LAL reagent helps to estimate the endotoxin in the sample.

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019)
This video ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Biochemical assays

Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations

Source: Neun, B. W. et al., <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/58830">Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays.</a> J. Vis. Exp. (2019).
This video describes a gel-clot assay to determine the presence of bacterial endotoxins in nano-formulations using Limulus Amoebocyte Lysate derived from the blood cells of horseshoe crabs. The assay helps test for endotoxin contamination of pharmaceutical products, including nano-based formulations.

Source: Neun, B. W. et al., Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays. J. Vis. Exp. (2019).
This video ...

An Assay for TLR-Dependent NF-&kcy;B/AP-1 Transcription Factor Signaling in Macrophages

Source: McKiel, L. A., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60317">A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to examine the toll-like receptor(TLR)-dependent activity of the transcription factors nuclear factor-kappa-B (NF-&#954;B) and activator protein-1 (AP-1) in macrophages. The TLRs trigger a pro-inflammatory signaling cascade upon recognizing damage-associated molecular patterns (DAMPs), activating NF-&#954;B and AP-1. The engineered reporter macrophages produce secreted embryonic alkaline phosphatase (SEAP) enzyme upon NF-&#954;B and AP-1 activation, which is detected via a chromogenic reaction.

1. Media and Reagent Preparation

	Prepare fibroblast media. Combine 450 mL of Dulbecco&#39;s modified Eagle medium (DMEM), 50 mL of fetal bovine serum ...

Immunology

Immunodiagnostics

Specialized Assays

An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition

Centrifuge 20 volumes of the peptide-GNP hybrid solution in 1.5-milliliter eppendorf tubes at 18,000 times g for 30 minutes. Carefully discard the supernatants. Transfer the hybrids to a single fresh tube, and wash them twice with 1 milliliter of PBS. After this, resuspend the washed hybrids in one volume of R10 medium. Mix equal volumes of the concentrated hybrids and the LPS-containing R10 medium such that the final concentration of the hybrids and LPS are 100 hundred nanomolar and 10 nanograms per milliliter respectively.
Next, remove the culture medium from the macrophage culture plate. Add 100 microliters of the hybrid-LPS mixture into each well with three replicates for each condition. Include a negative control and an LPS control. Incubate at 37 degrees Celsius for 24 hours, then, transfer the medium of each well to a separate centrifuge tube. Centrifuge the tubes at 18,000 times g in 4 degrees Celsius for 30 minutes. Transfer the supernatants into a fresh 96-well round bottom plate.
To begin the reporter assay on these samples, add 180 microliters of pre-warmed QUANTI-Blue solution to each well of a new 96-well flat bottom plate, then transfer 20 microliters of the supernatants from each sample into this plate. Incubate at 37 degrees Celsius for one to two hours until the dark blue color develops to an optical density over 1. After this, use a plate reader to record the absorption at 655 nanometers. Next, transfer 10 microliters of fresh supernatant from each sample into a 96-well clear flat-bottom white plate.
Using an automatic injector, add 50 microliters of the luciferase solution to each well and immediately record the luminescence well by well. Then, validate the inhibitory effect of the potential candidates and evaluate the TLR specificity as outlined in the text protocol.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

Summary

Explore More Videos

Chapters in this video

Air-sampled Filter Analysis for Endotoxins and DNA Content

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A Macrophage Reporter Cell Assay to Examine Toll-Like Receptor-Mediated NF-kB/AP-1 Signaling on Adsorbed Protein Layers on Polymeric Surfaces

Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Evaluating the Immune Response of a Nanoemulsion Adjuvant Vaccine Against Methicillin-Resistant Staphylococcus aureus (MRSA) Infection

Turbidimetric Limulus Amoebocyte Lysate Assay: A Technique to Detect and Quantify Endotoxin Contamination in Nano-Formulated Drugs

Gel-Clot Limulus Amoebocyte Lysate Assay: A Method for Bacterial Endotoxin Detection in Nanoparticle Formulations

An Assay for TLR-Dependent NF-кB/AP-1 Transcription Factor Signaling in Macrophages

An Assay to Screen Bioactive Nanoparticles for Toll-Like Receptor Signaling Inhibition