Endotoxin Removal Kit
First Edition (Revised on April, 2016)
Bacterial endotoxin is the unique component of gram negative bacteria cell wall. It’s the exogenous pyrogen which could activate macrophages, neutrophils, etc. to release Inflammatory mediators, and further to cause injury of the body, septic shock or even death. As endotoxin is an important pollutant of biological products, it is crucial to strictly control endotoxin level in biological products and medicines, which have direct access to human and animals. Therefore, the removal of endotoxin is very important in downstream purification process of biological products.
Polymyxin is a group of peptide antibiotics produced by Bacilluspolymyxa. Pplymyxin B (PMB) can be used as antagonist of endotoxin, although there is controversy in the mechanism of interation of PMB and bacterial endotoxin, it’s commonly accepted that there aer electrostatic interaction, ionic reaction and hydrophilic interaction, etc. The kit is based on this principle, it removes endotoxin through the binding of PMB (in the affinity resin) and endotoxin.
The main characteristics of the affinity resin are listed as following：
|Norms||1.5 ml prepacked column|
|Binding Capacity||≥2,000,000 EU/ml resin|
|Matrix||4% cross-linked agarose gel|
|Particle Size||90 µm|
|Working Life||18 months|
|Equilibration Buffer||phosphate buffer, pH 8.0|
|Ion Condition||0.1-0.5 M NaCl|
|Tolerated Reagents||20% DMSO, 20% alcohol,20% glycerol,1M urea,300mM imidazole, 0.05% twain-20, 10 mM DTT, etc.|
REAGENTS AND MATERIALS PROVIDED
|Affinity Resin||1.5 ml prepacked column|
|Regeneration Buffer||250 ml|
|Equilibration Buffer||250 ml|
|Flow Rate Controller||1|
|No Pyrogen Receiving Tubes||1 bag (3 tubes/bag)|
|No Pyrogen Tips(1 Ml)||2 bags (6 tips/bag)|
*The binding effect won’t be influenced for 5 times.
REAGENTS AND MATERIALS NEED TO BE PREPARED BY CUSTOMERS
1. Column Holder
2. 0.1M NaOH and 0.1M HCl to adjust the PH.
3. NaCl and Alcohol
STORAGE AND PERIOD OF VALIDITY
Store at 4℃. The Period of Validity is 1 year.
Endotoxin removal of proteins, peptides, antibodies, etc. The final Endotoxin level would be less than 0.1 EU/ml.
1. Sample Preparation: Adjust the concentration of NaCl in samples within 0.15-0.5M with no endotoxin 5M, and adjust the PH to 7-8.
2. Resin Activation: Assemble the prepacked column in vertical, remove the top cover, open the flow rate controller and get the protection solution drain. Then adding 5 ml regeneration buffer, keep the flow rate at 0.25ml/min (or10 drops/min) until drain, then add 5ml regeneration buffer again and repeat twice to get rid of endotoxins in system. (Note: This step needs to be performed even the first-time use)
3. Balance Resin: Add 6ml equilibration buffer, adjust the flow controller and maintain the flow rate at 0.5ml/min, drain the equilibration buffer. Repeat twice.
4. Endotoxin removal: Switch off the flow rate controller, and assay samples with no pyrogen tips. After assaying, open the controller and keep the flow rate at 0.25ml/min, collect samples when the effluent reaches 1.5 ml. Then adding 1.5-3.0ml equilibration buffer to wash when samples drain, and collect the fluid. At last, detect the concentration and endotoxin level of the sample. (If it doesn’t reach expected value, it needs to regenerate the resin according to step 2.)
5. Column Store: After experiments, equilibrate the column with 10 ml equilibration buffer. Add 1.5 ml regeneration buffer (contains 0.02% NaN3) after equilibration buffer drains. Then store the column at 2‐8℃.
1. Please strictly control the operating environment of the experiment to avoid outside endotoxin.
2. Once the tip box opened, tips need to be used one time.
|Removal efficiency is low||PH is not suitable||Adjust the PH to 7-8|
|Incubation time of samples and affinity resin is too short||Lower the flow rate of samples, or incubate at low temperature|
|Contamination happens in removal or detection systems||Use no pyrogen materials/reagents|
|Strong binding within endotoxin and proteins||1. Adjust pH|
2. Lower the flow rate of samples
|High sample loss||Non-specific binding effect within samples and affinity resin.||Increase the NaCl content in samples and equilibration buffer.|
|Strong binding within endotoxin and protein||1. Adjust pH|
2. Lower the flow rate of samples
|Sample has been contaminated||Different samples dealt with same column||Avoid using same prepacked column with different samples. If several samples need to be processed, you could wash the resin with 10-20 ml 2M NaCl before deal with another sample.|
|Regeneration buffer appear cloudy||The regeneration buffer maybe cloudy at room temp||Cool the regeneration buffer on the ice or regenerate at 4℃|