Patent ID: 8877456

Claim:
A method for determining the quantity of a botulinum neurotoxin expressed in LD 50 units/ml in a sample comprising a quantity of the botulinum neurotoxin, wherein the method comprises the following steps: (i) selecting a muscle tissue capable of being electrically stimulated to induce the contraction of the muscle tissue, and determining the minimum voltage V m needed to induce the contraction of muscle tissue, the muscle tissue being immersed in a buffer and connected to an electrical stimulator through a motor nerve; (ii) adding the sample containing a quantity of the botulinum neurotoxin having a concentration of 0 to 100 LD 50 units/ml; (iii) electrically stimulating, at a voltage at least equal to V m , the muscle tissue at certain time intervals by train pulse electrical stimulations thereby extending the life span of the muscle tissue, allowing long testing, or reducing muscle fatigue, wherein each stimulation period is for a time t S and is separated from the next stimulation period by a period lasting a time t P during which no stimulation is exerted, wherein the ratio t S /t P is from 1:2 to 1:50,000; (iv) measuring the effect induced by the sample and the effect induced by varying concentrations of a reference substance, wherein the effect is the variation in the force of contraction of the muscle tissue and wherein said reference substance is a botulinum neurotoxin type A; and (v) comparing the effect induced by the sample to the effect induced by said varying concentrations of the reference substance and thereby determining the quantity of the botulinum neurotoxin in the sample; wherein the method is carried out for at least 24 hours for data capture and until a reduction of 90% of the force of contraction of the muscle tissue is measured, thereby increasing the sensitivity of the method.