Cell-based method for determining an activity of botulinum toxin

Abstract

A new cell line and an antibody for determining the activity of botulinum toxin are disclosed. Also disclosed is a method of determining the activity of botulinum toxin using the cell line and/or the antibody.

1 . A method for determining activity of botulinum toxin in a sample, comprising the steps of:
 (a) contacting a cell with the sample, wherein the cell is from a cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106), wherein the clonally derived cell line has homogenous cell population, and wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell under same condition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell under same condition, and no sensitivity to BoNT/E of 10-400 pM concentration;   (b) obtaining cell lysate of the cell of (a), said cell lysate comprising proteins of the cell of (a) or isolating proteins from the cell of (a);   (c) contacting the cell lysate or the isolated proteins with an agent which specifically binds synaptosomal nerve-associated protein 25 (SNAP25 FL ) or botulinum toxin-cleaved SNAP25 fragment (SNAP25 197 );   (d) detecting the presence of a complex between the agent and the SNAP25 FL  and/or SNAP25 197 , and   (e) determining the activity of botulinum toxin in the sample, wherein the higher the amount of the agent-antigen SNAP25 FL  and/or SNAP25 197  complex detected the higher the level of botulinum toxin activity in the sample.   
     
     
         2 . The method of  claim 1 , wherein the botulinum toxin is botulinum toxin type A (BoNT/A). 
     
     
         3 . The method of  claim 1 , which further comprises, prior to step (a),
 culturing the cell in a culture medium supplemented with ganglioside GT1b trisodium salt (GT1b).   
     
     
         4 . The method of  claim 3 , wherein the culture medium further comprises creatine and arginine. 
     
     
         5 . The method of  claim 3 , wherein the concentration of GT1b is 25-75 μg/ml. 
     
     
         6 . The method of  claim 4 , wherein concentration of arginine is about 5 mM. 
     
     
         7 . The method of  claim 1 , wherein the agent is an antibody comprises:
 a heavy-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 11 to 13, 28 to 33, and 55 to 56;   a heavy-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 14 to 16, 34 to 39, and 57 to 58;   a heavy-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs. 17 to 19, 40 to 46, and 59 to 60;   a light-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62;   a light-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and   a light-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to 66.   
     
     
         8 . A method for detecting botulinum toxin in a sample, comprising the steps of:
 (a) contacting a cell with the sample comprising botulinum toxin or suspected of comprising botulinum toxin, wherein the cell is from a cell line clonally derived from parental neuro-2a cell (accession number KCTC AC28106), wherein the clonally derived cell line has homogenous cell population, and wherein the clonal cell line comprises cells susceptible to intoxication by botulinum toxin type A (BoNT/A) by about 25 pM or less of BoNT/A, and the cell shows higher sensitivity to BoNT/A, BoNT/B, BoNT/C, and BoNT/F compared to the parental neuro-2a cell under same condition, equal sensitivity to BoNT/D of 5 pM or 200 pM concentration to the parental neuro-2a cell under same condition, and no sensitivity to BoNT/E of 10-400 pM concentration;   (b) obtaining cell lysate of the cell of (a), said cell lysate comprising proteins of the cell of (a), or isolating proteins from the cell of (a);   (c) contacting the cell lysate or the isolated proteins with an agent which specifically binds synaptosomal nerve-associated protein 25 (SNAP25 FL ) or botulinum toxin-cleaved SNAP25 fragment (SNAP25 197 );   (d) detecting the presence of a complex between the agent and the SNAP25 FL  and/or SNAP25 197 ; and   (e) determining that when agent-antigen SNAP25 FL  and/or SNAP25 197  complex is detected, the botulinum toxin is present in the sample.   
     
     
         9 . The method of  claim 8 , wherein the botulinum toxin is botulinum toxin type A (BoNT/A). 
     
     
         10 . The method of  claim 8 , which further comprises, prior to step (a),
 culturing the cell in a culture medium supplemented with ganglioside GT1b trisodium salt (GT1b).   
     
     
         11 . The method of  claim 10 , wherein the culture medium further comprises creatine and arginine. 
     
     
         12 . The method of  claim 10 , wherein the concentration of GT1b is 25-75 μg/ml. 
     
     
         13 . The method of  claim 11 , wherein concentration of arginine is about 5 mM. 
     
     
         14 . The method of  claim 8 , wherein the agent is an antibody comprises:
 a heavy-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 11 to 13, 28 to 33, and 55 to 56;   a heavy-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 14 to 16, 34 to 39, and 57 to 58;   a heavy-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs. 17 to 19, 40 to 46, and 59 to 60;   a light-chain CDR1 region which is any one selected from the group consisting of SEQ ID NOs: 20 to 22, 47 to 49, and 61 to 62;   a light-chain CDR2 region which is any one selected from the group consisting of SEQ ID NOs: 23 to 24, 50 to 51, and 63 to 64; and   a light-chain CDR3 region which is any one selected from the group consisting of SEQ ID NOs: 25 to 27, 52 to 54, and 65 to 66.