endotoxin activity assay: a chemiluminescence-based bioassay for investigating endotoxin activity in blood sample

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

First, place the EA tubes to be used in tube racks and remove the caps. Use a combi-pipette to transfer 1 milliliter of the EA reagent from the bottle into tubes number 1, number 2, and number 3, by pipetting down the side of the tubes. Gently invert the blood collection tube 20 times to mix the patient&#39;s blood sample. Then, pipette 0.5 milliliters of patient blood into tube number 4 and tube number 5. Vortex tube number 4 for 10 seconds.
Place the tube racks with all of the EA test tubes into the incubator shaker, close the lid, and incubate at 37 degrees Celsius for 10 minutes. After this, open the incubator lid, and remove the tube racks from the shaker.
Vortex tube number 5. Using a sterile tip, pipette 40 microliters of blood into tubes number 1 and number 2, in duplicate.
Vortex tube number 4. Using the same pipette tip, transfer 40 microliters of blood from tube number 4 into tube number 3, in duplicate.
Vortex the six final test tubes one by one. Place the tube racks back into the incubating shaker and close the lid. Set the shaker to 100 RPM and start the motion for 14 minutes.
Insert the EA-labeled chip card into the chemiluminometer and press &#39;Start.&#39; After the 14-minute incubation, follow the instructions displayed on the chemiluminometer. Gently vortex each tube for 10 seconds before placing it onto the sample holder of the chemiluminometer.
Open the sample drawer and place tube number 1 into the sample holder. Then, close the sample drawer and wait for the Relative Light Unit reading. Repeat this reading process for tubes 2 and 3, and all duplicates as outlined in the text protocol.
After all of the tubes have been processed, the EA results will be calculated and printed automatically.
Repeat the entire assay process for each blood sample that needs to be tested. Store the EA assay kit and its components in the original sealed package, at a temperature between 2 and 25 degrees Celsius.

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Endotoxin Activity Assay (EAA)
<ol>
	<li>Collect patient blood samples in sterile tubes containing EDTA anticoagulant. Store blood samples at room temperature</li>
	<li>Prepare the EA test tubes for each patient&#39;s blood sample you need to test. Put the tubes in tube racks. Then remove the caps.</li>
	<li>Each EA test consists of 5 different kinds of tubes; use each one for a different portion of the test (refer next steps).
	<ol>
		<li>Use tube #1 (the &#34;Control&#34; tube) to measure the basal activity of the non-specific oxidative burst of patient&#39;s neutrophils in the absence of a specific antibody.</li>
		<li>Use tube #2 (the &#34;Sample&#34; tube) to measure the oxidative burst in response to the Lipopolysaccharide&#160;(LPS)-antibody complex.</li>
		<li>Use tube #3 (the &#34;Max&#34; tube) to measure the maximal oxidative burst of patient&#39;s neutrophils in response to an excess of endotoxin.</li>
		<li>Use tube #4 (the &#34;LPS&#34; tube) as a source of exogenous endotoxin.</li>
		<li>Use tube #5 (&#34;Aliquot&#34; tube) for blood storage. 
		NOTE:&#160;Duplicates of tubes #1, #2, and #3 are provided for a total of 8 tubes to be used for each blood sample being tested (the EA reagent bottle and quality control test can be used for all the tests contained in a pouch).</li>
	</ol>
	</li>
	<li>Using a combi-pipette, pipette a 1 mL volume of the EA reagent from the bottle into tubes #1 (Control tube), #2 (Sample tube), and #3 (Max tube), each one in duplicate. 
	NOTE:&#160;Pipette down the side of the tube to avoid solution splashing back up.</li>
	<li>Mix the patient blood sample by gently inverting the blood collection tube 20 times. Then, pipette 0.5 mL of patient blood into tube #4 (LPS max tube) and tube #5 (Aliquot tube). Vortex tube #4 for 10 s.</li>
	<li>Put the tube racks with all the EA test tubes in the incubator shaker. Close the lid and incubate for 10 min at the temperature of 37&#176;C.</li>
	<li>Open the lid and remove the tube racks from the incubator shaker. Vortex tube #5 (Aliquot tube). Using a sterile tip, pipette 40 &#181;L of blood into tubes #1 and #2, in duplicate.</li>
	<li>Vortex tube #4 (LPS tube). Using the same pipette tip, pipette 40 &#181;L of blood from tube #4 into tube #3 (Max tube), in duplicate.</li>
	<li>Vortex the six final test tubes (#1, #2, #3, and respective duplicates), then place them back into their racks. 
	NOTE:&#160;Ensure that all the tubes are vortexed for the same amount of time.</li>
	<li>Put the tube racks back into the incubating shaker and close the lid. Set the incubating shaker at 100 rpm, then start the motion for 14 min.</li>
	<li>Insert the EA-labeled chip card in the chemiluminometer and press start. After the 14 min incubation, follow the instructions displayed on the chemiluminometer to read the EA tubes in the correct order.</li>
	<li>Gently vortex each tube for 10 s before placing it onto the sample holder of the chemiluminometer. Open the sample drawer and place tube #1 in the sample holder. Then, close the sample drawer and wait for the Relative Light Unit (RLU) reading.</li>
	<li>Repeat step 1.12 for tube 2 and tube 3.</li>
	<li>Repeat step 1.12 for duplicate tubes 1, 2, and 3. 
	NOTE:&#160;Try to vortex all tubes for the same amount of time during steps 1.12&#8211;1.14.</li>
	<li>After all the tubes have been processed, note that the EA results will be calculated and printed automatically. Levels are expressed as EA units and represent the mean of duplicate determinations from the same samples.</li>
	<li>Repeat steps 1.4 to 1.15 for every blood sample that needs to be tested.</li>
	<li>Once the assay has been completed, store the remaining test tubes and EA reagents at 2&#8211;8 &#176;C for up to 30 days.</li>
</ol>

<table><tbody><tr><td>EAA kit</td><td>Spectral Medical Inc.</td><td>EAAST-20</td><td>Package with 20 tests + 1 quality control</td></tr><tr><td>Smart Line TL</td><td>Berthold</td><td>EAASL</td><td>Luminometer</td></tr><tr><td>Incubator shaker</td><td>GRANT</td><td>ES-20</td><td>Mini-incubator shaker</td></tr><tr><td>Vortexer</td><td>VWR</td><td>444-2790</td><td>Vortex instrument</td></tr></tbody></table>

Source: Pinciroli, R. et al. <a href="https://www.jove.com/v/58507">Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients.</a> J. Vis. Exp. (2019).
This video demonstrates a rapid bioassay for investigating endotoxin activity in a whole blood sample via a chemiluminescence-based method. This assay is a rapid and sensitive biomarker test for early-stage diagnosis of endotoxin-mediated sepsis.

Source: Pinciroli, R. et al. Endotoxin Activity Assay for the Detection of Whole Blood Endotoxemia in Critically Ill Patients. J. Vis. Exp. (2019).
This ...

In Gram-negative bacteria, lipopolysaccharide endotoxins are vital components of the bacterial outer membrane.
Structurally, an endotoxin is comprised of the O-antigen - outermost polysaccharide chain connected via a central core to lipid A, an immunostimulatory glycolipid crucial for endotoxin activity.
To investigate endotoxin activity, take a test tube with a suitable buffer containing anti-lipid A antibodies, zymosan - an exogenous neutrophil activator, and luminol - a chemiluminescent agent.
Suspend an optimum volume of an infected blood sample comprising endotoxins and blood cells, including neutrophils. Incubate the tube under physiological conditions.
During incubation, anti-lipid A antibody recognizes the lipid-A component of the endotoxin and binds to it via antigen-binding domains, resulting in the formation of an antibody-endotoxin complex.
 The endotoxin-bound antibody&#39;s FC domain binds to the neutrophils&#39; complementary receptor, presenting the attached endotoxins to neutrophils. This binding acts as a priming stimulus for neutrophils, triggering their activation.
Activated neutrophils engulf the zymosan and initiate an immune response, resulting in a rapid release of reactive oxygen species, ROS, from neutrophils. These ROS facilitate luminol oxidation, resulting in light emission by chemiluminescence.
The chemiluminescence signal intensity corresponds to the endotoxin activity in the blood sample.

91 vues. Dosage de l’activité des endotoxines : un essai biologique basé sur la chimiluminescence pour l’étude de l’activité des endotoxines dans un échantillon de sang

Vidéo: Dosage de l’activité des endotoxines : un essai biologique basé sur la chimiluminescence pour l’étude de l’activité des endotoxines dans un échantillon de sang - Expérience

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample

Transcription

Endotoxin Activity Assay: A Chemiluminescence-Based Bioassay for Investigating Endotoxin Activity in Blood Sample