Endotoxin Removal Kit

Instruction manual

First Edition (Revised on April, 2016)

PRODUCT INFORMATION

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.

PRINCIPLE

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
Ligand	modified PMB
Phrange	pH 5-10
Matrix	4% cross-linked agarose gel
Particle Size	90 µm
Storage Temperature	2-8°C
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

Name	Quantity
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)
Instruction Manual	1

*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.

APPLICATIONS

Endotoxin removal of proteins, peptides, antibodies, etc. The final Endotoxin level would be less than 0.1 EU/ml.

ASSAY PROCEDURES

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℃. 

IMPORTANT NOTES

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.3. For your safety and health, please wear a lab-gown and disposable gloves in experiments.

TROUBLE SHOOTINGS

Problems	Possible reasons	Solutions
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℃