Method for quantification of neurotoxin
View Patent ↗The present invention relates to a method for quantitatively measuring the muscular relaxing activity of a neurotoxin. Specifically, based on an extent of the activity of muscular relaxation of a neurotoxin from bacteria of Clostridium , the present invention relates to a method for quantification of the efficacy (potential and/or diffusion reaction) of a neurotoxin comprising the following steps of: (a) administering a neurotoxin to the hind leg muscle of one of hind legs of a non-human mammal; (b) applying electric stimulus to said non-human mammal; (c) measuring a compound muscle action potential (CMAP) by contraction of said hind leg muscle to which the neurotoxin is administered and/or of the hind leg muscle of the other hind leg to which the neurotoxin is not administered; and (d) taking amplitude data from the compound muscle action potential (CMAP) obtained by the measurement in step (c) and analyzing an extent of a decrease in amplitude to thereby quantify the efficacy of the muscular relaxing activity by the neurotoxin. In contrast to the mouse LD 50 currently used as a potential unit of a botulinum toxin which is measurable at a level of only several units, the quantification method of the efficacy of a neurotoxin of the present invention allows for measurement at a level of as low as 0.01 to 1 unit and hence is a method with a high sensitivity, reproducibility and accuracy.
1. A method for quantification of the efficacy of a neurotoxin, comprising the following steps of:
(a) administering a neurotoxin to the hind leg muscle of one of hind legs of a non-human mammal;
(b) applying electric stimulus to said non-human mammal;
(c) measuring a compound muscle action potential (CMAP) by contraction of said hind leg muscle to which the neurotoxin is administered and/or of the hind leg muscle of the other hind leg to which the neurotoxin is not administered; and
(d) taking amplitude data from the compound muscle action potential (CMAP) obtained by the measurement in step (c) and quantifying the efficacy of the muscular relaxing activity by the neurotoxin with calibration curves obtained by statistical analysis of the amplitude of CMAP.
2. The method for quantification of claim 1 wherein, in step (c), a compound muscle action potential (CMAP) by contraction of the hind leg muscle to which the neurotoxin is administered is measured.
3. The method for quantification of claim 1 wherein said efficacy is a potential of the neurotoxin.
4. The method for quantification of claim 1 wherein, in step (c), a compound muscle action potential (CMAP) by contraction of the hind leg muscle of the other hind leg to which the neurotoxin is not administered is measured.
5. The method for quantification of claim 1 wherein said efficacy is a diffusion reaction of the neurotoxin.
6. The method for quantification of claim 1 wherein, in step (c), a compound muscle action potential (CMAP) by contraction of both the hind leg muscle to which the neurotoxin is administered and the hind leg muscle of the other hind leg to which the neurotoxin is not administered is measured simultaneously to thereby simultaneously quantify both a potential and a diffusion reaction of the neurotoxin.
7. The method for quantification of claim 1 wherein an extent of a decrease in amplitude of a compound muscle action potential (CMAP) by the neurotoxin is analyzed in step (d) above.
8. The method for quantification of claim 1 wherein said statistical analysis is carried out by regression analysis or Logit transformation.
9. The method for quantification of claim 1 wherein a non-human mammal needs not be surgically treated for said measurement.
10. The method for quantification of claim 1 wherein the neurotoxin is selected from a neurotoxin from bacteria, of Clostridium , a neurotoxin produced by fish and shellfish, or a neurotoxin derived from a snake toxin.
11. The method for quantification of claim 10 wherein the neurotoxin is one from bacteria of Clostridium selected from Clostridium baratii, Clostridium butyricum, Clostridium botulinum , or Clostridium tetani.
12. The method for quantification of claim 11 wherein the neurotoxin is one from Clostridium botulinum.
13. The method for quantification of claim 1 wherein difference in the efficacy of two or more different neurotoxins is quantitatively compared.
14. The method for quantification of claim 1 , wherein, in steps (b) and (c), clip electrodes are used to apply the electric stimulus and to measure the CMAP.