International Journal of Food Microbiology, 11 (1990) 271-278
271
Elsevier FOOD 00338
Assay in mice for low levels of Clostridium botulinum toxin M o t o h i d e Takahashi, Shoichi K a m e y a m a and Genji Sakaguchi 1 Department of Applied Immunology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo and I College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka, Japan (Received 1 February 1990; accepted 28 May 1990)
When botulinum toxin at a low level such as 0.1 to 1.0 mouse intraperitoneal LDso was injected subcutaneously into a mouse at the inguinocrual region, abdominal ptosis with local palsy developed. If this symptom is taken as a marker, 1.0 mouse intraperitoneal LDs0 can be detected within 6 h and 0.1 LDs0 within 24 h. The severity of symptoms and the time-to-death in days after injection of toxin were converted into scores to quantify the toxic activity. Over a wide range of dose, between 0.075 and 38.4 mouse intraperitoneal LDs0, a linear relationship was obtained between the log dose and the score. By use of this method, low levels of toxin such as 0.1 mouse intraperitoneal LDs0 can be titrated accurately and easily. Key words: Clostridium botulinum toxin; In vivo titration; Subcutaneous injection; Inguinocrual region; Abdominal ptosis
Introduction
Botulism is caused by botulinum neurotoxin, which is undoubtedly the most potent bacterial toxin known. The toxin can be titrated by various methods as listed in Table I. Of the various in vitro methods, the most sensitive one is amplified ELISA (Shone et al., 1986) which detects 9 mouse intraperitoneal LDs0 (hereafter referred to as LDs0) of toxin. Of the various in vivo methods, the intramuscular injection method (Sugiyama et al., 1975) is the most sensitive, detecting 0.1 LDs0 of toxin. Improvement of the assay method with respect to the sensitivity, simplicity, and rapidity is strongly required in view of the rather rapid course of the illness. In addition, strabismus, blephalospasm, and other disorders due to dystonia have recently been treated by microinjection of botulinum toxin (Scott, 1980; Carruthers, 1985), therefore more sensitive and accurate assay methods are urgently required to titrate low levels of toxin for human therapeutic use. Correspondence address: Motohide Takahashi, Department of Applied Immunology, National Institute of Health, 2-10-35 Kamiosaki, Shinagawa-ku, Tokyo 141, Japan. 0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
272 TABLE I Sensitivities of different methods reported for assaying botulinum toxin Method
Sensitivity a
Reported by
In vitro b RIA RIA RPHA RPHA RPLA ELISA ELISA ELISA CLISA
100 MLD 80 LD5o 10 LDso 1000 LDso 50 LDso 50 LDso 9 LD5o 400 LDso 20 MLD
Boroff and Chu-Chen, 1973 Ashtom Crowther and Dolly, 1985 Sakaguchi et al., 1974 Sonnenschein, 1978 Horiguchi, Kozaki and Sakaguchi, 1984 Notermans, Dufrenne and van Schothorst, 1978 Shone et al., 1986 Kozaki et al., 1979 Ligieza et al., 1986
In vivoc IP IV 1V IM
1 LDso 1000 LDso 500 LDso 0.1 LDso
Sakaguchi, Sakaguchi and Imai, 1964 Boroff and Fleck, 1966 Sakaguchi, Sakaguchi and Kondo, 1968 Sugiyama, Brenner and DasGupta, 1975
a The lowest detectable level in mouse intraperitoneal LDs0 or MLD. b RIA, Radio immunoassay; RPHA, Reversed passive hemagglutination; RPLA, Reversed passive latex agglutination; ELISA, Enzyme-linked immunosorbent assay; IP, Intraperitoneal injection; IV, Intravenous injection; IM, Intramuscular injection. c In mice.
The present investigation was carried out to develop a n assay m e t h o d for low levels of b o t u l i n u m toxin. We noticed that palsy of the a b d o m i n a l muscle hitherto n o t described develops after s u b c u t a n e o u s injection of a low level of b o t u l i n u m toxin into the m o u s e at the i n g u i n o c r u a l region. T h e severities of s y m p t o m s were converted to scores a n d statistical analysis of these scores established an assay m e t h o d for titration of low levels of the toxin.
Materials and Methods The toxin C. botulinum type A strain 97 was g r o w n in P Y G m e d i u m consisting of 2% p e p t o n e ( M i k u n i K a g a k u , Tokyo), 0.5% yeast extract (Oriental Yeast, Osaka), 1% glucose, 0.01% cysteine at p H 7.0 for 4 days at 3 0 ° C . T h e toxin was partially purified b y acid precipitation at p H 3.5, salting out at 40% saturated a m m o n i u m sulfate, a n d gel filtration o n Sephadex G-200 ( P h a r m a c i a F i n e Chemicals, U p p s a l a , Sweden). The p r e p a r a t i o n c o n t a i n e d a specific toxicity of 1.0 × 10 8 L D s 0 / m g nitrogen. T h e toxicity was d e t e r m i n e d by i n t r a p e r i t o n e a l injection of mice with serial dilutions of a sample.
273
Subcutaneous injection into mice
The toxin was diluted two-fold serially in 0.2% gelatin-0.05 M phosphate buffered saline, pH 6.3. Each dilution in 0.2-ml doses was injected subcutaneously at the inguinocrual region into 10 mice (strain ddY-Shizuoka, female) weighing 18 to 22 g. The mice were observed for 4 days after injection. The symptoms were converted statistically into scores and toxin titer was estimated by the score method (Ipsen, 1955; Kondo et al., 1959; Sakaguchi et al., 1968). Neutralization test
For neutralization of the toxin, horse type A antitoxin given by the Chiba Serum Institute, Ichikawa-shi, Chiba, was used.
Results S y m p t o m s and conversion to scores
After subcutaneous injection of 0.2 LDs0 of toxin into the mouse, palsy localized to the abdominal muscle developed and the injection site became flaccid. When the mouse was hung down by holding the tail, the flaccid portion became protuberant so that the change was easily seen (Fig. 1). The symptoms in mice developed after injection of 0.075 to 38.4 LDs0 of the toxin were classified into the following six categories: (i) + : Local palsy of the abdominal muscle. (ii) + + : In addition to that of +, hypotonic abdomen noticeable by touching.
Fig. 1. Local palsy of the abdominal muscle in the mouse. The photograph was taken 24 h after injection of toxin of 0.1 LDs0 (Severity of the symptoms: + ).
274 TABLE II Scores given on the bases of severities of symptoms covering a 3-day period after subcutaneous injection of botulinum toxin On day a
Outcome
Severity of symptoms
Score
Dead Dead Dead Dead Dead Dead Survived Survived Survived Survived Survived Survived
+ + + + + to + + + b + + to + b +++++ c ++++ c + + + to + c
4.2 3.5 3.0 2.7 2.5 2.5 2.3 2.1 1.6 1.1 0.6 0
+ + + + + -
++++ +++ ++ +
a The day of injection was counted as day O. b Six hours after injection. c On day 1.
(iii) + + + : W a s p - s h a p e d a b d o m e n due to the h y p o t o n i c i t y a c c o m p a n i e d occasionally with l a b o u r e d breathing. (iv) + + + + : M a r k e d generalized weakness a n d flaccid paralysis of the leg of the injected side. (v) + + + + + : A b a s i a a n d l a b o u r e d breathing. (vi) Death: Most mice died within 3 days a n d only a few mice after 3 days. The s y m p t o m s were recorded for 3 days after injection, a n d a p p r o p r i a t e scores were given to the s y m p t o m s so that a linear relationship w o u l d be a t t a i n e d b e t w e e n the log dose a n d the symptoms. Care was t a k e n so that the scores were suitable for statistical analysis. The scores given i n response to the s y m p t o m s are s h o w n in T a b l e II. Eleven graded scores were given, from 0 (no s y m p t o m s ) to 4.2 (death).
Dose-response curve T h e results of statistical analysis are s h o w n in Fig. 2. The linearity of the dose response curve was observed b e t w e e n doses from - 7 . 9 (0.075 LDs0 ) to - 5 . 2 (38.4 LDso ). H o m o g e n e i t y of variance a m o n g the doses was d e n i e d ( P = 0.05), a n d the c o m m o n variance (s 2) calculated from these results was 0.0635.
Time-related changes of the score T o x i n i n three different doses (0.15, 2.4 a n d 38.4 LD50 ) was injected into mice a n d the s u b s e q u e n t changes in the score at various times are shown in Fig. 3. I n the figure, the average scores of 10 mice at the different doses were plotted. I n the group injected with 38.4 LDs0 , the score was 0.06 i n a n hour, a n d it increased gradually,
275
o 2
O
uo
5X
I
i
|
/
-8
-7
-6
-5
Dose in l o g Fig. 2. Dose-response curve of C. botulinum toxin. Geometric means of scores of 10 mice were plotted versus the log doses in dilution factors.
5
4
3 0J
u 2 to
1
1
t
2
I
5 10 20 Time(h ) a f t e r i n j e c t i o n
I
50
I
100
I
200
Fig. 3. Time-related changes of the score. Vertical bars are the 95% confidence intervals. ©, 38.4 LDso; e, 2.4 LDso; % 0.15 LDso.
276 reaching a plateau at a score of 4.2 in 24 h. In the group injected with 0.15 LDs0, the appearance of symptoms was retarded and the score was 0.6 in 24 h and reached a plateau at a score of 0.95 in 48 h as was the case with the 2.4 LDs0-injected group.
Discussion Various methods of titration of botulinum toxin have been reported. In diagnosis of botulism, it seems essential to titrate the direct toxicity in a bioassay system, and a more sensitive and reliable method is needed. The method used routinely to detect and titrate the toxin is the mouse intraperitoneal injection method. By this method, toxin down to 2 to 3 LDs0 can be titrated (Sakaguchi et al., 1964). As the method depends on such discrete variables as death and survival of the mice, many mice are needed for titration of a sample and the range of the straight line relationship is rather narrow. It was reported that type E toxin can be titrated with a fiducial limit of about 30% within 1 to 2 h by intravenous injection into mice and the use of the score method (Sakaguchi et al., 1968). The lowest detectable level by this method, however, was about 500 LDs0/ml. Intramuscular injection of the toxin into the leg of the mouse and using local palsy as a marker, in contrast to the intravenous method, can detect as small a dose as 0.1 LDs0 (Sugiyama et al., 1975). Their method is based upon injection of the toxin into a specific muscle, musculus gastrocnemius (calf muscle). It it not a simple technique to use routinely. The present work attempts to establish a method for titration of low levels of botulinum toxin by injecting the toxin through a different route. By subcutaneous injection at the inguinocrual region, palsy localized to the abdominal muscle, a hitherto unrecognized symptom, was observed. The palsy was considered to be due to flaccid paralysis. When such a mouse was hung by holding the tail, ptosis of the abdomen was easily observable (Fig. 1). Such a symptom was completely suppressed by neutralization with the specific antitoxin. The symptoms became strongest within 3 days (Fig. 3). When the mice were observed for an additional 7 to 14 days, the symptoms, particularly mild ones, were lessened (results not included). It seems, therefore, justified to read the results within 3 days after injection in the proposed method. This method differs from the one based upon such discrete variables as death and survival in that detailed information of each mouse can be used in the test. In fact, in the present investigation, a wide range from 0.075 to 38.4 LDs0, gave a linear dose-response curve (Fig. 2). This method requires fewer animals and furnishes higher reproducibility than the method using discrete variables. When abdominal ptosis is utilized, toxin of 0.1 LDs0 can be detected in 24 h.
Acknowledgements The authors wish to thank Dr. S. Ishida, Chief, Laboratory Biological Products, Department of General Biologics Control, National Institute of Health, Tokyo, for his helpful advice on statistical analysis.
277
References Ashton, A.C., Crowther, J.S. and Dolly, J.O. (1985) A sensitive and useful radio immuno-assay for neurotoxin and its haemagglutinin complex from Clostridium botulinurn. Toxicon 23, 235-246. Boroff, D.A. and Chu-Chen, G. (1973) Radio immuno-assay for type A toxin of Clostridium botulinum. Appl. Microbiol. 25, 545-549. Boroff, D.A. and Fleck, U. (1966) Statistical analysis of a rapid in vivo method for the titration of the toxin of Clostridiurn botulinurn. J. Bacteriol. 92, 1580-1581. Carruthers, J.A. (1985) Ophthalmologic use of botulinum A exotoxin. Can. J. Ophthalmol. 20, 135-141. Horiguchi, Y., Kozaki, S. and Sakaguchi, G. (1984) Determination of Clostridium botulinum toxin by reversed passive latex agglutination. Jpn. J. Vet. Sci. 46, 487-491. lpsen, J. (1955) Appropriate scores in bio-assay using death-times and survivor symptoms. Biometrics 11, 465 -480. Kondo, H., Kondo, S., Hirose, S., Kurokawa, M., Ishida, S. and Fujisaki, Y. (1959) The assay of diphtheria toxin using mouse. Jpn. J. Med. Sci. Biol. 12, 331-342. Kozaki, S., Dufrenne, J., Hagenaars, A.M. and Notermans, S. (1979) Enzyme-linked immunosorbent assay (ELISA) for detection of Clostridium botulinum type B toxin. Jpn. J. Med. Sci. Biol. 32, 199-205. Ligieza, J., Michalik, M., Reiss, J. and Grzybowski, J. (1986) Determination of soluble antigens of Clostridium botulinum A by chemiluminescent-immunosorbentassay (CLISA). Arch. Immunol. Ther. Exp. 34, 189-195. Notermans, S., Dufrenne, J. and van Schothorst, M. (1978) Enzyme-linked immunosorbent assay for detection of Clostridium botulinurn toxin type A. Jpn. J. Med. Sci. Biol. 31, 81-85. Sakaguchi, G., Sakaguchi, S. and Imai, N. (1964) Comparative gel filtration of toxin precursor and trypsin-activated toxin of Clostridium botulinum type E. J. Bacteriol. 87, 401-407. Sakaguchi, G., Sakaguchi, S. and Kondo, H. (1968) Rapid bioassay for Clostridiurn botulinurn type E toxins by intravenous injection into mice. Jpn. J. Med. Sci. Biol. 21, 369-378. Sakaguchi, G., Sakaguchi, S., Kozaki, S., Sugii, S. and Ohishi, I. (1974) Cross reaction in reversed passive hemagglutination between Clostridiurn botulinum type A and B toxins and its avoidance by the use of anti-toxic component immunoglobulin isolated by affinity chromatography. Jpn. J. Med. Sci. Biol. 27, 161-172. Scott, A.B. (1980) Botulinum toxin injection into extraocular muscles as an alternative to strabismus surgery. Opthalmology 87, 1044-1049. Shone, C., Wilton-Smith, P., Appleton, N., Hambleton, P., Modi, N., Gatley, S. and Melling, J. (1986) Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay. Appl. Environ. Microbiol. 50, 63-67. Sonnenschein, B. (1978) Use of the reversed passive hemagglutination in detection of Clostridium botulinum type A, B and E toxin. Zbl. Bakteriol. Hyg. I Abt. Orig. A 240, 221-234. Sugiyama, H., Brenner, S.L. and DasGupta, B.R. (1975) Detection of Clostridium botulinum toxin by local paralysis elicited with intramuscular challenge. Appl. Microbiol. 30, 420-423.
271
Elsevier FOOD 00338
Assay in mice for low levels of Clostridium botulinum toxin M o t o h i d e Takahashi, Shoichi K a m e y a m a and Genji Sakaguchi 1 Department of Applied Immunology, National Institute of Health, Kamiosaki, Shinagawa-ku, Tokyo and I College of Agriculture, University of Osaka Prefecture, Sakai-shi, Osaka, Japan (Received 1 February 1990; accepted 28 May 1990)
When botulinum toxin at a low level such as 0.1 to 1.0 mouse intraperitoneal LDso was injected subcutaneously into a mouse at the inguinocrual region, abdominal ptosis with local palsy developed. If this symptom is taken as a marker, 1.0 mouse intraperitoneal LDs0 can be detected within 6 h and 0.1 LDs0 within 24 h. The severity of symptoms and the time-to-death in days after injection of toxin were converted into scores to quantify the toxic activity. Over a wide range of dose, between 0.075 and 38.4 mouse intraperitoneal LDs0, a linear relationship was obtained between the log dose and the score. By use of this method, low levels of toxin such as 0.1 mouse intraperitoneal LDs0 can be titrated accurately and easily. Key words: Clostridium botulinum toxin; In vivo titration; Subcutaneous injection; Inguinocrual region; Abdominal ptosis
Introduction
Botulism is caused by botulinum neurotoxin, which is undoubtedly the most potent bacterial toxin known. The toxin can be titrated by various methods as listed in Table I. Of the various in vitro methods, the most sensitive one is amplified ELISA (Shone et al., 1986) which detects 9 mouse intraperitoneal LDs0 (hereafter referred to as LDs0) of toxin. Of the various in vivo methods, the intramuscular injection method (Sugiyama et al., 1975) is the most sensitive, detecting 0.1 LDs0 of toxin. Improvement of the assay method with respect to the sensitivity, simplicity, and rapidity is strongly required in view of the rather rapid course of the illness. In addition, strabismus, blephalospasm, and other disorders due to dystonia have recently been treated by microinjection of botulinum toxin (Scott, 1980; Carruthers, 1985), therefore more sensitive and accurate assay methods are urgently required to titrate low levels of toxin for human therapeutic use. Correspondence address: Motohide Takahashi, Department of Applied Immunology, National Institute of Health, 2-10-35 Kamiosaki, Shinagawa-ku, Tokyo 141, Japan. 0168-1605/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
272 TABLE I Sensitivities of different methods reported for assaying botulinum toxin Method
Sensitivity a
Reported by
In vitro b RIA RIA RPHA RPHA RPLA ELISA ELISA ELISA CLISA
100 MLD 80 LD5o 10 LDso 1000 LDso 50 LDso 50 LDso 9 LD5o 400 LDso 20 MLD
Boroff and Chu-Chen, 1973 Ashtom Crowther and Dolly, 1985 Sakaguchi et al., 1974 Sonnenschein, 1978 Horiguchi, Kozaki and Sakaguchi, 1984 Notermans, Dufrenne and van Schothorst, 1978 Shone et al., 1986 Kozaki et al., 1979 Ligieza et al., 1986
In vivoc IP IV 1V IM
1 LDso 1000 LDso 500 LDso 0.1 LDso
Sakaguchi, Sakaguchi and Imai, 1964 Boroff and Fleck, 1966 Sakaguchi, Sakaguchi and Kondo, 1968 Sugiyama, Brenner and DasGupta, 1975
a The lowest detectable level in mouse intraperitoneal LDs0 or MLD. b RIA, Radio immunoassay; RPHA, Reversed passive hemagglutination; RPLA, Reversed passive latex agglutination; ELISA, Enzyme-linked immunosorbent assay; IP, Intraperitoneal injection; IV, Intravenous injection; IM, Intramuscular injection. c In mice.
The present investigation was carried out to develop a n assay m e t h o d for low levels of b o t u l i n u m toxin. We noticed that palsy of the a b d o m i n a l muscle hitherto n o t described develops after s u b c u t a n e o u s injection of a low level of b o t u l i n u m toxin into the m o u s e at the i n g u i n o c r u a l region. T h e severities of s y m p t o m s were converted to scores a n d statistical analysis of these scores established an assay m e t h o d for titration of low levels of the toxin.
Materials and Methods The toxin C. botulinum type A strain 97 was g r o w n in P Y G m e d i u m consisting of 2% p e p t o n e ( M i k u n i K a g a k u , Tokyo), 0.5% yeast extract (Oriental Yeast, Osaka), 1% glucose, 0.01% cysteine at p H 7.0 for 4 days at 3 0 ° C . T h e toxin was partially purified b y acid precipitation at p H 3.5, salting out at 40% saturated a m m o n i u m sulfate, a n d gel filtration o n Sephadex G-200 ( P h a r m a c i a F i n e Chemicals, U p p s a l a , Sweden). The p r e p a r a t i o n c o n t a i n e d a specific toxicity of 1.0 × 10 8 L D s 0 / m g nitrogen. T h e toxicity was d e t e r m i n e d by i n t r a p e r i t o n e a l injection of mice with serial dilutions of a sample.
273
Subcutaneous injection into mice
The toxin was diluted two-fold serially in 0.2% gelatin-0.05 M phosphate buffered saline, pH 6.3. Each dilution in 0.2-ml doses was injected subcutaneously at the inguinocrual region into 10 mice (strain ddY-Shizuoka, female) weighing 18 to 22 g. The mice were observed for 4 days after injection. The symptoms were converted statistically into scores and toxin titer was estimated by the score method (Ipsen, 1955; Kondo et al., 1959; Sakaguchi et al., 1968). Neutralization test
For neutralization of the toxin, horse type A antitoxin given by the Chiba Serum Institute, Ichikawa-shi, Chiba, was used.
Results S y m p t o m s and conversion to scores
After subcutaneous injection of 0.2 LDs0 of toxin into the mouse, palsy localized to the abdominal muscle developed and the injection site became flaccid. When the mouse was hung down by holding the tail, the flaccid portion became protuberant so that the change was easily seen (Fig. 1). The symptoms in mice developed after injection of 0.075 to 38.4 LDs0 of the toxin were classified into the following six categories: (i) + : Local palsy of the abdominal muscle. (ii) + + : In addition to that of +, hypotonic abdomen noticeable by touching.
Fig. 1. Local palsy of the abdominal muscle in the mouse. The photograph was taken 24 h after injection of toxin of 0.1 LDs0 (Severity of the symptoms: + ).
274 TABLE II Scores given on the bases of severities of symptoms covering a 3-day period after subcutaneous injection of botulinum toxin On day a
Outcome
Severity of symptoms
Score
Dead Dead Dead Dead Dead Dead Survived Survived Survived Survived Survived Survived
+ + + + + to + + + b + + to + b +++++ c ++++ c + + + to + c
4.2 3.5 3.0 2.7 2.5 2.5 2.3 2.1 1.6 1.1 0.6 0
+ + + + + -
++++ +++ ++ +
a The day of injection was counted as day O. b Six hours after injection. c On day 1.
(iii) + + + : W a s p - s h a p e d a b d o m e n due to the h y p o t o n i c i t y a c c o m p a n i e d occasionally with l a b o u r e d breathing. (iv) + + + + : M a r k e d generalized weakness a n d flaccid paralysis of the leg of the injected side. (v) + + + + + : A b a s i a a n d l a b o u r e d breathing. (vi) Death: Most mice died within 3 days a n d only a few mice after 3 days. The s y m p t o m s were recorded for 3 days after injection, a n d a p p r o p r i a t e scores were given to the s y m p t o m s so that a linear relationship w o u l d be a t t a i n e d b e t w e e n the log dose a n d the symptoms. Care was t a k e n so that the scores were suitable for statistical analysis. The scores given i n response to the s y m p t o m s are s h o w n in T a b l e II. Eleven graded scores were given, from 0 (no s y m p t o m s ) to 4.2 (death).
Dose-response curve T h e results of statistical analysis are s h o w n in Fig. 2. The linearity of the dose response curve was observed b e t w e e n doses from - 7 . 9 (0.075 LDs0 ) to - 5 . 2 (38.4 LDso ). H o m o g e n e i t y of variance a m o n g the doses was d e n i e d ( P = 0.05), a n d the c o m m o n variance (s 2) calculated from these results was 0.0635.
Time-related changes of the score T o x i n i n three different doses (0.15, 2.4 a n d 38.4 LD50 ) was injected into mice a n d the s u b s e q u e n t changes in the score at various times are shown in Fig. 3. I n the figure, the average scores of 10 mice at the different doses were plotted. I n the group injected with 38.4 LDs0 , the score was 0.06 i n a n hour, a n d it increased gradually,
275
o 2
O
uo
5X
I
i
|
/
-8
-7
-6
-5
Dose in l o g Fig. 2. Dose-response curve of C. botulinum toxin. Geometric means of scores of 10 mice were plotted versus the log doses in dilution factors.
5
4
3 0J
u 2 to
1
1
t
2
I
5 10 20 Time(h ) a f t e r i n j e c t i o n
I
50
I
100
I
200
Fig. 3. Time-related changes of the score. Vertical bars are the 95% confidence intervals. ©, 38.4 LDso; e, 2.4 LDso; % 0.15 LDso.
276 reaching a plateau at a score of 4.2 in 24 h. In the group injected with 0.15 LDs0, the appearance of symptoms was retarded and the score was 0.6 in 24 h and reached a plateau at a score of 0.95 in 48 h as was the case with the 2.4 LDs0-injected group.
Discussion Various methods of titration of botulinum toxin have been reported. In diagnosis of botulism, it seems essential to titrate the direct toxicity in a bioassay system, and a more sensitive and reliable method is needed. The method used routinely to detect and titrate the toxin is the mouse intraperitoneal injection method. By this method, toxin down to 2 to 3 LDs0 can be titrated (Sakaguchi et al., 1964). As the method depends on such discrete variables as death and survival of the mice, many mice are needed for titration of a sample and the range of the straight line relationship is rather narrow. It was reported that type E toxin can be titrated with a fiducial limit of about 30% within 1 to 2 h by intravenous injection into mice and the use of the score method (Sakaguchi et al., 1968). The lowest detectable level by this method, however, was about 500 LDs0/ml. Intramuscular injection of the toxin into the leg of the mouse and using local palsy as a marker, in contrast to the intravenous method, can detect as small a dose as 0.1 LDs0 (Sugiyama et al., 1975). Their method is based upon injection of the toxin into a specific muscle, musculus gastrocnemius (calf muscle). It it not a simple technique to use routinely. The present work attempts to establish a method for titration of low levels of botulinum toxin by injecting the toxin through a different route. By subcutaneous injection at the inguinocrual region, palsy localized to the abdominal muscle, a hitherto unrecognized symptom, was observed. The palsy was considered to be due to flaccid paralysis. When such a mouse was hung by holding the tail, ptosis of the abdomen was easily observable (Fig. 1). Such a symptom was completely suppressed by neutralization with the specific antitoxin. The symptoms became strongest within 3 days (Fig. 3). When the mice were observed for an additional 7 to 14 days, the symptoms, particularly mild ones, were lessened (results not included). It seems, therefore, justified to read the results within 3 days after injection in the proposed method. This method differs from the one based upon such discrete variables as death and survival in that detailed information of each mouse can be used in the test. In fact, in the present investigation, a wide range from 0.075 to 38.4 LDs0, gave a linear dose-response curve (Fig. 2). This method requires fewer animals and furnishes higher reproducibility than the method using discrete variables. When abdominal ptosis is utilized, toxin of 0.1 LDs0 can be detected in 24 h.
Acknowledgements The authors wish to thank Dr. S. Ishida, Chief, Laboratory Biological Products, Department of General Biologics Control, National Institute of Health, Tokyo, for his helpful advice on statistical analysis.
277
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