EAA™ ENDOTOXIN ACTIVITY ASSAY
EAA™ ENDOTOXIN ACTIVITY ASSAY
The Endotoxin Activity Assay (EAA™) is the only commercially available test to measure endotoxin activity in whole blood that is cleared by the US Food and Drug Administration (FDA), licensed by Health Canada in 2003, and CE marked.
Until the EAA™, there has been no reliable method to measure endotoxin accurately in the bloodstream. It is an semi-quantitative test in the measurement of endotoxin activity (EA) and allows for rapid measurements whereby results can be obtained in approximately 30 minutes.
ENDOTOXIN AND SEPSIS
Endotoxin is a major component of the cell wall of Gram Negative bacteria and is an important microbial mediator of sepsis. The EAA™ can be used as a diagnostic tool for measuring endotoxin activity and allows patients to be stratificatied based on endotoxin load.
Therefore, the utility of the EAA™ as a diagnostic tool, when used in conjunction with relevant diagnostic tests and clinical information, may allow for risk assessment of patients for progression to severe sepsis based on EA measurement.
A prospective cohort study, MEDIC (Multi-Center Endotoxin Detection in Critical Illness) of 857 consecutive new patients to ICU evaluated the usefulness of EAA™ as a diagnostic tool in sepsis and septic shock. Specifically, the study was carried out in order to determine the prevalence of endotoxemia in the critically ill as well as to determine if any association exists between EAA™ values at time of admission to ICU and disease outcome. Results of the study indicate that rate of endotoxemia was quite high in critically ill patients: 26.6% of the cohort had EA levels 0.4-0.6 and 30.6% of the cohort had EA levels ≥0.6. Further analyses showed that for the 74 patients meeting the criteria for severe sepsis, EA levels at the time of ICU admission were significantly higher (0.57±0.26 vs. 0.46±26, P<0.001).
Multivariate regression analysis of the data uncovered that EA levels at time of admission had a significant association with the development of severe sepsis on day of ICU admission, either as a continuous variable (P=0.02) or as a categorical one (P=0.0001); when patients were stratified based on EA levels to low (<0.4), intermediate (0.4- 0.6) and to high (>0.6) groups, it was noted that the odds of developing severe sepsis in the first 24 hrs of ICU admission was 2.0 (95% CI) for the intermediate group and 3.0 (95% CI) for the high group. These data strongly indicated the usefulness of EAA™ as a diagnostic tool in initial risk stratification and subsequent management of septic patients.
EAA™ ENDOTOXIN ACTIVITY ASSAY
The Endotoxin Activity Assay (EAA™) is the gold standard for measuring Endotoxin Activity in human whole blood, and has been utilized in human and veterinary research projects around the world.
The EAA™ is a chemiluminescent bio-assay based on the oxidative burst reaction of activated neutrophils to complement coated LPS-IgM immune complexes.
The IgM antibody, a key reagent, is specific for the Lipid A portion of endotoxin (LPS). In the presence of LPS, the ensuing oxidative burst results in light emission in the presence of luminol.
Each EAA™ test relies on 3 reactions: the first reaction is a negative control which allows each patient to be their own control, the second is the test sample and the third is a maximum chemiluminescence calibrator with a high level of exogenous endotoxin. The maximum calibrator is necessary as endotoxins from different species of Gram negative bacteria have a slightly different reactivity in the EAA™. E. coli O55:B5 is used as the standard as it has a mid-level reactivity in the assay.
The EAA™ is a highly sensitive test for endotoxin activity with the greatest sensitivity in the lowest concentration ranges of endotoxin. The dose response curve flattens in the higher endotoxin activity ranges, with no hook effect observed.
The EAA™ is a semi-quantitative test that will provide an Endotoxin Activity (EA) result. The endotoxin activity result will stratify patients into a low (<0.40), intermediate (0.40-0.59) and high (≥0.60) endotoxin activity level, allowing for stratification of patients based on endotoxin load.
The EAA dose response is an exponential curve characterized by two asymptotes. The EAA generates a unit less number, called endotoxin activity, which correlates to the concentration of endotoxin in the sample.
The graphic represents dose response for E. coli O55:B5 endotoxin. The shape of the curve changes slightly depending on the species of bacteria the endotoxin is derived from. Further, Spectral is available to provide a variety of EAA reagents to research customers in order to allow customization of the assay to specific research needs, such as different animal studies. For more information and specific dose response information, contact us.
HOW THE EAA™ IS DIFFERENT*
LAL vs. EAA™
The EAA™ uses a highly specific IgM antibody for the most preserved structural component of endotoxin, Lipid-A. This allows the EAA™ to specifically measure endotoxin activity in human whole blood, something the Limulus Amebocyte Lysate (LAL) assay is not capable of doing.
The LAL assay is a highly sensitive test for endotoxin measurement in crystalloid solutions, and is not accurate when utilized in whole blood or plasma. This is due to the fact that blood contains many components known to inhibit or activate the enzymatic cascade of the LAL, such as serine proteases. In order to attempt to use the LAL for blood based endotoxin testing, the sample must first be treated with strongly acidic or caustic chemicals, increasing the risk to the user, complexity of the test, and assay time from sample to result.
The EAA™ is a rapid assay (approximately 30 minutes from sample to result) designed specifically for endotoxin activity measurement in whole blood.
*A rapid assay of endotoxin in whole blood using autologous neutrophil dependent chemiluminescence. Romaschin AD, Harris DM, Ribeiro MB, et al., J. Immunol. Methods 1998; 212: 169-185.
The EAA™ Endotoxin Activity Assay information and instructions for use video.
Spectral manufactures high quality endotoxin calibration kits aimed to enable a quantitative measurement of endotoxin concentration by means of the EAA™ method.
These calibrators are custom manufactured using endotoxin derived from the gram negative bacterial species, and concentration(s) of interest to the end user.
For inquiries and purchasing, please contact:
EAA™ ENDOTOXIN ACTIVITY ASSAY
How does the EAA™ detect endotoxin?
The EAA™ detects endotoxin by employing a highly specific IgM antibody with a strong affinity to the Lipid-A portion of endotoxin (Lipopolysaccharide, LPS).
What is the difference between EAA™ and the LAL assay?
The EAA™ is a semi quantitative test for endotoxin activity in whole blood. The LAL is a quantitative test for endotoxin in colloidal solutions. The LAL cannot reliably and reproducibly detect endotoxin in whole blood, plasma, or serum.
What is the sensitivity of the EAA™?
The analytical sensitivity for the EAA™ was estimated from the precision observed in a low EAA™ level sample. The sensitivity was calculated based on extrapolation of the observed standard deviation in the low level sample to a “zero” EAA™ level. The overall EAA™ mean was 0.106 with a standard deviation of 0.021 EA units. The analytical sensitivity can be estimated by applying the standard deviation to a hypothetical measurement of “zero” EA units where the analytical sensitivity is equal to 2 times the SD or 0.042 EA units.
What is the specificity of the EAA™?
The EAA™ is specific to Gram-Negative Lipopolysaccharide (Endotoxin). The EAA™ does not detect gram positive bacterial, or yeast cell wall components as no reactivity is observed when lipoteichoic acid extracts up to 2000 pg/mL are tested for the following strains of Gram positive bacteria: S. mutans, S. pyogenes, S. sanguis, S. faecalis, S. aureus and B. subtilis. Similarly, there was no reactivity of yeast mannan up to 2000 pg/mL and cell wall extracts of C. albicans and A. fumagatis.
Where does specificity of the EAA™ for LPS come from?
The specificity of the EAA™ is due to the highly specific IgM antibody used for the detection of endotoxin.
What is the range of the EAA™?
The EAA™ will provide an endotoxin activity result normally between 0.0 and 1.0. The EA result correlates with a low (0.0-0.39), intermediate (0.4-0.59) or high (≥0.60) endotoxin activity level.
What is the LLD/LOD of the EAA™?
The analytical sensitivity of the EAA™ is 0.042 EA units. This is approximately 20-50 pg/ml of endotoxin, depending on bacterial species of origin and neutrophil response.
What is the molecular form of the endotoxin that the assay detects?
The EAA™ detects antibody-complexed endotoxin in the disaggregated form.
In which animal species is the EAA™ known to work, in which species is it known not to work?
The intended use of the EAA™ is to measure endotoxin activity in Human whole blood. However, there are publications available demonstrating the assay’s utility in measuring endotoxin activity in Horses and Dogs. There are ongoing unpublished studies utilizing the EAA™ in measuring endotoxin activity in Pigs. The EAA™ cannot be used to determine the Endotoxin Activity in Mice as the antibody contained in the assay is Murine IgM.
What specimens can be used with EAA™?
The sample specimen must be whole blood drawn via indwelling catheter or venipuncture. The sample can be stored at room temperature (18-25OC) in an EDTA anticoagulated draw tube, and must be used to run the EAA™ within 3 hours of draw.The sample specimen must be whole blood drawn via indwelling catheter or venipuncture. The sample can be stored at room temperature (18-25OC) in an EDTA anticoagulated draw tube, and must be used to run the EAA™ within 3 hours of draw.