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

Overview

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.

Protocol

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)

1. Collect patient blood samples in sterile tubes containing EDTA anticoagulant. Store blood samples at room temperature
2. Prepare the EA test tubes for each patient's blood sample you need to test. Put the tubes in tube racks. Then remove the caps.
3. Each EA test consists of 5 different kinds of tubes; use each one for a different portion of the test (refer next steps).
   1. Use tube #1 (the "Control" tube) to measure the basal activity of the non-specific oxidative burst of patient's neutrophils in the absence of a specific antibody.
   2. Use tube #2 (the "Sample" tube) to measure the oxidative burst in response to the Lipopolysaccharide (LPS)-antibody complex.
   3. Use tube #3 (the "Max" tube) to measure the maximal oxidative burst of patient's neutrophils in response to an excess of endotoxin.
   4. Use tube #4 (the "LPS" tube) as a source of exogenous endotoxin.
   5. Use tube #5 ("Aliquot" tube) for blood storage.
      NOTE: 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).
4. 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: Pipette down the side of the tube to avoid solution splashing back up.
5. 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.
6. 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°C.
7. Open the lid and remove the tube racks from the incubator shaker. Vortex tube #5 (Aliquot tube). Using a sterile tip, pipette 40 µL of blood into tubes #1 and #2, in duplicate.
8. Vortex tube #4 (LPS tube). Using the same pipette tip, pipette 40 µL of blood from tube #4 into tube #3 (Max tube), in duplicate.
9. Vortex the six final test tubes (#1, #2, #3, and respective duplicates), then place them back into their racks.
   NOTE: Ensure that all the tubes are vortexed for the same amount of time.
10. 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.
11. 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.
12. 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.
13. Repeat step 1.12 for tube 2 and tube 3.
14. Repeat step 1.12 for duplicate tubes 1, 2, and 3.
    NOTE: Try to vortex all tubes for the same amount of time during steps 1.12–1.14.
15. 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.
16. Repeat steps 1.4 to 1.15 for every blood sample that needs to be tested.
17. Once the assay has been completed, store the remaining test tubes and EA reagents at 2–8 °C for up to 30 days.

Materials

Name	Company	Catalog Number	Comments
EAA kit	Spectral Medical Inc.	EAAST-20	Package with 20 tests + 1 quality control
Smart Line TL	Berthold	EAASL	Luminometer
Incubator shaker	GRANT	ES-20	Mini-incubator shaker
Vortexer	VWR	444-2790	Vortex instrument