Dear Dr.

Detecting endotoxin in plasma is problematic.  Clinical samples tested with the rabbit fever test have demonstrated the ability to inactivate the pyrogenic properties of endotoxin (1,2).  Though some of this inhibition is due to enzymatic activity, the majority is due to complex formation between endotoxin and molecules in the blood (2).  Several specific components in the blood have been shown to affect endotoxin detection with the Limulus Ameobocyte Lysate (LAL) or recombinant Factor C (rFC) assays:

• Serine proteses (3).
• Amidases (3).
• Bilirubin (3).
• Esterases (4).
• Phosphatases (5,6) 
• Lipoproteins (7).
• Cationic proteins (e.g. lysozyme, IgG, hemoglobin) (8).
• Lactoferrin (9).
• Platelets (10,11).

Plasma samples have been shown to neutralize up to 95% of endotoxin activity, have 500-fold variations when confirmed with ELISA assays, give false positive rates over 10%, and produce recovery rates less than 30% (12-16).

The most prevalent strategy to overcome these difficulties is to use a heat and dilution protocol.  This can often work when testing endotoxin in pharmaceutical or laboratory samples.  However, heat-treatment alone is insufficient with plasma (17-20).  As shown above, many of the interfering proteins are not enzymatic and not affected by heat.  Given the negative charge and hydrophobic nature of endotoxin, many of the interactions require dilutions in excess of 1:1000 making detection of low-level endotoxin impossible.  Also, heat-treatment alone can actually make endotoxin detection worse due to:

• Morphological changes in fibrinogen (3)
• Altered endotoxin-lipoprotein interaction (4).
• Physical changes to platelets (10).
• Altered interactions with kinetic and chromogenic LAL tests (4).

Because of these difficulties we have developed the ESP (Endotoxin Sample Preparation) Kit for detection of endotoxin in blood plasma samples.

ESP combines heat-inactivation, pH shift, and enzymatic degradation in specially designed buffers to produce plasma samples that contain negligible amounts of interfering components.

• ESP has no effect on endotoxin activity.
• ESP treatment typically requires less than 60 minutes.
• ESP requires modest sample dilution for minimum detection of 0.1-1.0 EU/ml.
• ESP produces routine sample endotoxin recovery in excess of 75%.
• ESP routinely provides Positive Product Control (PPC) recovery over 75%.

•  Treatment results:
• 10 citrated plasma samples, triplicate testing:
• LPS recovery increased from <5% to 73-92%.
• PPC recovery changed from 163-183% to 87-91% (i.e. the level of inaccuracy decreased from 63-83% to 9-13%).

• The extent of protein removal with ESP is shown in the PAGE analysis:
• The first lane is untreated plasma.  The second lane is the same plasma treated with ESP. 

Look for the release of BioDtech's ESP Endotoxin Sample Preparation Kit this Summer!