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...

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>Turbidity LAL Assay</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>Chromogenic LAL Assay</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>Gel-Clot LAL Assay</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 ...

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 ...

detection-endotoxin-nano-formulations-using-limulus-amoebocyte-lysate

Research

JoVE Journal

Bioengineering

14.4K 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

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...