Non-diffusive botulinum toxin causing local muscle paralysis, and purification method thereof
The present invention relates to a method for purifying a non-spreading botulinum toxin that causes local muscle paralysis and a non-spreading botulinum toxin obtained thereby. The method comprises the steps of: subjecting a purified botulinum toxin type A product to ion-exchange chromatography using a controlled pH of buffer, concentration of sodium chloride (NaCl), thereby separating the botulinum toxin type A product into subfractions; and collecting a subfraction having an A260/A280 value in a specific range from the separated subfractions.
1. A method for determining a non-spreading botulinum toxin, comprising:
injecting the non-spreading botulinum toxin into the either left or right hind limb calf muscle of mouse (4-6 wk old, weighing 18-22 g) in an amount equivalent to 1.5-3 times the LD50 of the toxin in the volume ranges of 20-50 μL; and
assessing paralysis conditions of the hind limb muscles and respiratory muscles of the injected mouse and a survival rate of the injected mouse to determine a non-spreading botulinum toxin.
2. The method of claim 1 , wherein the non-spreading botulinum toxin shows a survival rate at least 80 % at 96 hours after injection of 2 U toxin preparation in the volume 50 μL either left or right hind limb calf muscle of the mouse.
3. The method of claim 2 , wherein the paralysis conditions comprises:
limb appearance and walking of normal mice;
mice walk with dragging, but toes are not put together;
mice walk with dragging, toes are put together;
mice walk with dragging, toes are put together, foot joints are bent inward; and
mice walk with dragging, toes are put together, foot joints are bent inward, feet come into contact with leg muscles, and legs are lame.