Removal of endotoxin is one of the most difficult downstream processes during protein purification. EndoTrap® red, as part of the EndoClear kit, is developed to meet the most challenging requirements of endotoxin removal: the highest endotoxin removal capacity combined with excellent recovery rates. EndoTrap® red is based on affinity chromatography for column or batch mode. The working range of EndoTrap® red is pH 6-9 and it works with an ionic strength up to 250 mM NaCl (<100 mM is recommended). Proteins, peptides and antibodies can be applied onto the EndoTrap® red column. LPS from Klebsiella pneumoniae and Serratia marcescens can only be removed with EndoTrap® red.
Besides the EndoTrap® red, EndoTrap® blue is also available. EndoTrap® blue has a pH range of 4-9 and is capable to work with an ionic strength up to 600 mM. Endotoxin in plasmid can only be removed with EndoTrap® blue.
The detection of endotoxin is based on studies of Frederick Bang. He observed that bacteria caused intravascular coagulation in the American horseshoe crab, Limulus polyphemus. In collaboration, Levin and Bang found that the agent responsible for the clotting phenomena resided in the crab's amoebocytes, or circulating blood cells, and that pyrogen triggers the turbidity and gel-forming reaction enzymatically.
The Mini-LAL assay for endotoxin detection, the second part of the EndoClear kit, makes use of an activated enzyme. In the presence of a colorless substrate, the enzymatic reaction will cause a yellow colour to develop upon cleavage of the chromophore, p-nitroaniline (pNA). The reaction is stopped by the addition of acetic acid and the absorbance at 405 nm is measured with a spectrophotometer. The endotoxin concentration of samples with unknown concentrations, which are run concurrently with the standards, can be determined from a standard curveThe Profos EndoTrap® red assay is intended for the removal of endotoxin with removal rates of >95% per cycle. The binding capacity of EndoTrap® red is 2,000,000 EU/ml (1EU = 100 pg LPS)
The Hycult Biotech Mini-LAL assay is intended for the quantitative detection of endotoxin in culture medium, buffers, and other solutions. The kit has a minimum endotoxin detection limit of 0.01 EU/ml and a measurable concentration range of 0.01 to 10 EU/ml.- Binding capacity of 2,000,000 EU/ml.
- Endotoxin removal rates of >95% per cycle.
- Final LPS concentration 0.005 - 0.1 EU/ml.
- Column or batch mode.
- Operating at pH 6.0-9.0.
- Suitable for proteins, peptides and antibodies.
- Suitable for an ionic strength up to 250 mM NaCl (< 100 mM is recommended).
- Suitable for LPS from Klebsiella pneumoniae and Serratia marcescens.
Hycult Biotech Mini-LAL assay (endotoxin detection):
- Minimum concentration which can be measured is 0.01 EU/ml.
- Measurable endotoxin detection range of 0.01-10 EU/ml.
- Working volume of 50 µl/well.
- 72 determinations
- Operating at pH 6.5-8.0.
- The Profos EndoTrap® red Assay is based on affinity chromatography with a working time of ~30 minutes.
- Sample is applied to the column with equilibration buffer.
- Fractions are collected.
- Endotoxin removal rate is >95% per cycle.
- Samples are measured with Hycult Biotech LAL Assay.
Hycult Biotech Mini-LAL assay (endotoxin detection):
- The LAL assay is an enzyme-based assay with a working time of 45 minutes.
- Samples and standards are incubated with LAL reagent.
- The enzymatic reaction, triggered by endotoxin, will cause a yellow color to develop upon cleavage of the chromophore, p-nitroaniline.
- The enzymatic reaction is stopped by the addition of acetic acid.
- The absorbance at 405 nm is measured with a spectrophotometer. A standard curve is obtained by plotting the absorbance (linear) versus the corresponding concentrations of the E. coli standards (log).
- The endotoxin concentration of samples, which are run concurrently with the standards, can be determined from the standard curve.
The Profos EndoTrap® red columns should be stored in regeneration buffer with 0.02% sodium azide.
After reconstitution the Hycult Biotech Mini-LAL assay reagents (E. coli standard and LAL reagent) should be stored in aliquots at -20 °C.- Gonçalves, S et al; Associations between renal function, volume status and endotoxaemia in chronic kidney disease patients. Nephrol Dial Transpl 2006, 21: 2788
- Bowdish, D et al; The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004, 172: 3758
- Bublin, M et al; Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses. Vaccine 2007, 25: 8395
- Cordery, D et al; Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue. J Infect Dis 2007, 195: 905
- Gonçalves, S et al; Associations between renal function, volume status and endotoxaemia in chronic kidney disease patients. Nephrol Dial Transpl 2006, 21: 2788
- Bowdish, D et al; The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004, 172: 3758
- Bublin, M et al; Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses. Vaccine 2007, 25: 8395
- Cordery, D et al; Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue. J Infect Dis 2007, 195: 905
ENDOCLEAR
• Combination of the Profos EndoTrap® and the Hbt Mini-LAL assay
• Two sensitive and specific products to remove, detect and quantify bacterial endotoxin
• Profos EndoTrap®: Efficient removal of bacterial endotoxins
• Mini-LAL assay: Quantitative measurement of endotoxin in culture medium, buffers and other solutions
Specific features ENDOTRAP®
• High specific binding of endotoxins (broad range of bacterial strains)
• Binding capacity of 2,000,000 EU/ml (1 EU = 100 pg LPS)
• Final LPS concentration 0.005-0.1 EU/ml
• High efficiency: removal rates of > 95% per cycle by means of highly specific EndoTrap® ligands
• High recovery rates: minimized sample loss
• Fast and easy to use, no incubation
• Reliable endotoxin removal, widely independent of pH, ion strength and temperature
• No special buffers required for washing or binding
• Column & batch mode
• Sample volume 2-50 ml
• Two types of ligand: EndoTrap® blue and EndoTrap® red
Specific features Mini-LAL assay
• Minimum concentration which can be measured is 0.005 EU/ml
• Measurable concentration range of 0.01 - 10 EU/ml
• Operating at pH 6.5-8.0
• The LAL assay is an enzyme-based assay with a working time of 60 minutes
• Samples and standards are incubated with LAL reagent
• The enzymatic reaction, triggered by endotoxin, will cause a yellow color to develop upon cleavage of the chromophore, p-nitroaniline
• The endotoxin concentration of samples, which are run concurrently with the standards, can be determined from the standard curve
|
|
EndoTrap® blue |
EndoTrap® red |
|
pH |
pH 4-9 |
pH 6-9 |
|
Ionic strength |
Up to 600 mM NaCl |
Up to 250 mM NaCl We recommend < 100 mM NaCl |
|
Suitable with EDTA and other calcium chelator containing buffers |
No |
Yes |
|
Customer specific equilibration buffer has to be enriched with calcium |
Yes |
No |
|
PBS can be used as equilibration buffer |
Yes, when enriched freshly with 50-100 µM Ca2+ |
Yes |
Selected reading
1. Gonçalves, S et al; Association between renal function, volume status and endotoxaemia in chronic kidney disease patients. Nephrol Dial Transpl 2006, 21: 2788
2. Bowdish, D et al; The human cationic peptide LL-37 induces activation of the extracellular signal-regulated kinase and p38 kinase pathways in primary human monocytes. J Immunol 2004, 172: 3758
3. Beauvillain, C et al; Neutrophils efficiently cross-prime naive T cells in vivo. Blood 2007, 110: 2965
4. Birkenmeier, G et al; Polymyxin B conjugated Alpha-2 Macroglobulin as an adjunctive therapy to sepsis: Modes of action and impact on lethality. J. Pharmacol.Exp. Ther. 2006, 318: 762
5. Bublin, M et al; Use of a genetic cholera toxin B subunit/allergen fusion molecule as mucosal delivery system with immunosuppressive activity against Th2 immune responses. Vaccine 2007, 25: 8395
6. Cordery, D et al; Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue. J Infect Dis 2007, 195: 905