APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1990,
p.
Vol. 56, No. 3
808-811
0099-2240/90/030808-04$02.00/0 Copyright © 1990, American Society for Microbiology
NOTES
Monoclonal Antibody to Type F Clostridium botulinum Toxin JOSEPH L. FERREIRA,'* MOSTAFA K. HAMDY,2 STEVEN G. McCAY,l AND FRANCIS A. ZAPATKA' Food and Drug Administration, Atlanta, Georgia 30309,1 and Food Science Department, University of Georgia, Athens, Georgia 306022 Received 20 September 1989/Accepted 18 December 1989
Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was -10 mouse lethal doses of toxin per ml of toxic fluid.
culture fluids were assayed by an indirect ELISA system for antibody against type F toxin. Selected antibody-producing hybridoma cells were subcloned by limiting dilution in 96-well microdilution plates, and antibody-producing clones were then chosen. Antibody-producing hybridomas were expanded and used in Pristane (Sigma)-primed mice by i.p. injection of 106 hybridoma cells to produce ascites. An indirect ELISA system similar to that described by Ferreira et al. (2) was used to screen for antibody-producing hybridomas. Type F toxoid consisting of 0.5 jig of protein in 100 [l of carbonate buffer (0.1 M, pH 9.6) was placed in each well of the microdilution plate (Immulon II, round bottom; Dynatech Laboratories, Inc., Alexandria, Va.) and held overnight at 4°C. After three washings of each well with buffer A (PBS [pH 7.4] containing 0.1% bovine serum albumin [BSA], 0.05% Tween 20, and 0.02% sodium azide), nonspecific binding sites were blocked with 1% BSA in buffer A for 1 h at 35°C. Plates were then washed three times with buffer A, and 100 ,ul of hybridoma culture supernatants was placed in each well and incubated for 2 h at 35°C. Plates were again washed as described above, and 100 jl of alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (IgG) conjugate (Sigma), diluted (1:1,000, vol/vol) in buffer A containing 1% BSA, was then added to each well. The plates were incubated at 35°C for 2 h and again washed four times as described above, and 100 [l of substrate (1 mg of p-nitrophenylphosphate per ml of glycine buffer [0.1 M, pH 10.4] containing 1 mM MgCl2 and ZnCl2) was added to each well. After 1 h of incubation at 35°C, the reaction was stopped by adding 25 jil of NaOH (3 M), and the A410 was measured spectrophotometrically. This basic assay was also used to examine normal mouse serum and ascites fluids by using type F toxin (WAKO Chemicals, Dallas, Tex.) and type F toxoid. A double-sandwich ELISA was used for the detection of type F toxin. In this procedure, ascites fluid from hybridoma 223 was diluted and dispensed into wells of a 96-well Dynatech microdilution plate. The plates were washed and then blocked by using 1% BSA. After the plates were washed, toxic culture supernatants, purified toxin preparations, or food samples were then added to each well. After sample incubation, plates were washed and rabbit polyclonal type F antitoxin (1.5 jig of protein per ml) was added to each
The first poisoning incident caused by type F Clostridium botulinum was observed in Denmark in 1958 (6). Type F toxin and other types of botulinum toxins can be detected by a variety of methods, including radioimmunoassay, gel diffusion, enzyme-linked immunosorbent assay (ELISA), and, currently the most sensitive procedure, the mouse bioassay (1, 7, 10, 11, 14). The lower sensitivities and specificities of the in vitro methods have been largely the result of the impurity of antigens used to produce the antibody (10). Monoclonal antibodies (MAbs) have been produced against toxin types A, B, C, D, and E of C. botulinum (2, 4, 5, 8, 12). In the present study, hybridomas were generated that produced MAbs against type F C. botulinum toxin. One MAb was used in combination with a double-sandwich ELISA system for the rapid detection of this toxin in foods, culture media, or other toxic preparations. BALB/c myeloma cell line Sp2/0-Ag-14NS(FO) was obtained from C. Aulosio, Centers for Disease Control, Atlanta, Ga., and was utilized for the production of type F hybridomas by fusion with spleen cells from an immunized BALB/c mouse. C. botulinum cultures (types A, B, C, D, E, and F), as well as cultures of other Clostridium spp. (C. sporogenes, C. novyi, and C. perfringens), were used to assess the specificity and sensitivity of the ELISA system. Type F C. botulinum toxin was isolated and purified by the method of Yang and Sugiyama (15). Female BALB/c mice were immunized as follows. Equal volumes of Freund complete adjuvant (Sigma Chemical Co., St. Louis, Mo.) were emulsified with F toxoid preparations in 0.06 M phosphatebuffered saline (PBS). The 0.5-ml emulsions, containing 100 jig of toxoid, were then injected intraperitoneally (i.p.) into mice. After 4 weeks, each mouse was again injected (i.p.) with 0.5 ml of toxoid (200 jug) in 0.06 M PBS. Three days after the second injection, the titers of the sera were measured and spleens were removed for fusion. Cells of the Sp2/0-Ag-14NS(FO) myeloma cell line used for fusion and type F hybridoma cells were grown in reinforced minimal essential medium (formula no. 78-5216; GIBCO Laboratories, Grand Island, N.Y.). The myeloma and spleen cells were fused at a 1:1 ratio by using 108 cells of each type following the procedure of Oi and Herzenberg (9). Tissue *
Corresponding author. 808
NOTES
VOL. 56, 1990
well. The plate was then washed, and alkaline phosphataselabeled goat anti-rabbit IgG conjugate (Sigma) was added. Again, after plates were washed, substrate solution (1 mg of p-nitrophenylphosphate per ml of glycine buffer [pH 10.4]) was added to each well. The reaction was then stopped, and the A410 was measured. The same procedure was also utilized with the rabbit polyclonal antibody serving as the first antibody and the hybridoma 223 MAb serving as the second antibody. The alkaline phosphatase-conjugated antimouse IgG and substrate were used as described above. To detect type F C. botulinum in foods, the Langeland strain was inoculated into oxygen-reduced food products (commercially canned liver pate, tuna fish, and crabmeat) and these products were incubated for 14 days at 32°C. Each product was then diluted (weight/volume) in PBS and assayed for the toxicity level (mouse lethal doses per milliliter) by using the mouse bioassay procedure of the Food and Drug Administration (3) and by the double-sandwich ELISA utilizing the type F MAb produced by hybridoma 223. Type F toxin from toxic culture fluid was also used to examine the neutralizing capacity of the MAb. A mixture of toxin and MAb was injected (i.p.) into Swiss mice, and the mice were observed for symptoms and death during 3 days of incubation. Isotyping was determined by an indirect ELISA. Antimouse sera (IgA, IgGl, IgG2a, IgG2b, IgG3, IgM [Sigma], kappa and lambda light chains [Miles Laboratories Inc., Elkhart, Ind.], IgD, and IgE [ICN Immuno-Biologicals, Lisle, Ill.]) were diluted 1:100 in 0.1 M carbonate buffer (pH 9.6) and refrigerated (4°C) overnight in Dynatech Immulon II microdilution plates. After plates were washed, mouse tissue culture fluids were incubated for 1 h at 24°C, plates were washed again, and alkaline phosphatase-labeled goat antimouse IgG (1:1,000 dilution) was added. After 1 h of incubation, the plates were washed and 1 mg of p-nitrophenylphosphate per ml of glycine buffer was added and incubated at 35°C for 30 min. The reaction was stopped by using 25 ,ul of 3 M NaOH per well, and the A410 was measured. Immuno-dot blots for toxin detection were prepared by diluting the WAKO toxin in PBS and 1 ,ul of toxin preparations and then were applied to nitrocellulose (NC) filters, allowed to dry for 10 min at 24°C, and blocked with PBS containing 0.3% Tween 20 (PBS-T) for 10 min. Ascites fluid, diluted 1:2,000 in PBS-T, was added to the NC and incubated for 1 h at 24°C. The NC was washed three times in PBS-T, and horseradish peroxidase-conjugated anti-mouse IgG (Sigma) (diluted 1:100 in PBS-T) was incubated with the NC for 1 h at 35°C. The NC was washed three times in PBS-T and then once with PBS before the substrate (0.5 mg of 3,3'-diaminobenzidine per ml of 0.003% H202 [Sigma]) was added. The substrate was incubated for 15 min, and the reaction was stopped by using fresh distilled water. MAb-producing hybridomas were generated against type F C. botulinum toxin. The titer of hybridoma 223 in ascites was assayed by the indirect ELISA system by using type F botulinum toxin (Fig. 1), and this analysis revealed a titer of _106/ml. Approximately 33% of the stable hybridomas produced IgM; the remainder synthesized IgGl or IgG2a. Figure 2 shows the double-sandwich ELISA results of various MAb dilutions (ascites of hybridoma 223), polyclonal type F mouse serum, and normal mouse serum against 0.5 ,ug of type F toxin per well. It is evident that the hybridoma 223 ascites titer is -100 times higher than that of polyclonal type F serum. The sensitivity of the doublesandwich ELISA system for the detection of type F toxin as a function of dilution (nanograms per milliliter) was observed to increase when the MAb rather than the polyclonal rabbit
809
0.4-
---
0.4--b
E 0 V-
0.3-
r_
S 0.2-\
0 .0 c .0 D0
.0
0.1
-
0.0
-
0 0
2
4 3
2
I
6 5
4
6
7
Log10 dilution of ascites fluids FIG. 1. Indirect ELISA assay of hybridoma 223 ascites fluid against 0.1 jig of C. botulinum type F toxin per well. Tenfold dilutions were tested and showed a titer of -106/ml.
serum was used as the first antibody (Fig. 3). No crossreactions were observed with other toxins of C. botulinum types A, B, or C, but the MAb did react slightly with toxin of C. botulinum type D. No reaction was observed with nontoxic C. novyi, C. perfringens, or C. sporogenes strains. However, type E reacted strongly when only undiluted culture supernatants were used (Table 1). When toxic foods were examined in the double-sandwich ELISA, toxicity was determined at levels equivalent to 10 mouse lethal doses per ml (Table 2). The immuno-dot blot examination of WAKO toxin was positive at a dilution of 5 ,ug/ml or a total of 5 ng of toxin on NC filters. The isotype of the hybridoma 223 MAb was determined to be IgG2a heavy chain and kappa light chain by the ELISA. Antibody produced by hybridoma 223 was chosen on the basis of its high titer and specificity for type F C. botulinum. We found that when the MAb was attached to the plate first this system gave added sensitivity over the rabbit serum (polyclonal) being used as the first antibody. This assay was also equally sensitive for detecting C. botulinum type F in culture fluids, foods, and toxic preparations made from type F toxin. Of 14 hybridomas selected from fusion plates for
1.6E o
1.2-
4
0.8
223 Ascites
0 0 .0
o
o
C U .0
Poly F
0.4
0.4-
0
Normal
0.0-1 I I 3
i
Log10 dilution
5
mouse
6
antibody
FIG. 2. Double-sandwich ELISA of mouse sera (normal and F polyclonal) and hybridoma 223 ascites fluid against 0.5 ,ug of type F Wako toxin per well. Tenfold dilutions were tested. The hybridoma 223 ascites titer is 100 fold greater than than of F polyclonal serum.
APPL. ENVIRON. MICROBIOL.
NOTES
810
TABLE 2. Detection of type F toxin in foods by using the double-sandwich ELISA and MAb 223
E
2
Mob 223
0 _E
°
0
c
aw C
°0
0
1
0
.0
.0
0
0 / 0-
O-
3
FIG. 3. Effect of first antibody (MAb from hybridoma 223 or rabbit polyclonal serum) on detection of type F Wako toxin in double-sandwich ELISA. The sensitivity increases when MAb rather than polyclonal rabbit serum is used as the first antibody.
expansion, only one (designated as hybridoma 223) was used in the double-sandwich ELISA system for toxin detection. This hybridoma was selected for its high titer and specificity for type F toxin. The MAb produced by hybridoma 223 used in the double-sandwich ELISA method did not neutralize type F toxin in 20-g mice. In the toxin molecule, there may be more than one epitope present and, since the MAb can react with one specific antigenic site or conformation, it is possible that other epitope(s) would be available to cause botulism in the mouse. It is conceivable that the MAb-toxin complex may dissociate in vivo following its injection. If the MAb has a low affinity for a particular toxin epitope, under certain conditions it would not protect the animal in toxin neutralization testing. Another possible explanation is that in similar but not identical antigenic determinants (epitopes) binding of the MAb occurs but with low affinity. MAbs TABLE 1. Cross-reaction of MAb 223 to different types and strains of C. botulinum and other clostridia by using double-sandwich ELISA Absorbance at a log1o dilution of:
Toxicity
(MLD/ml)a 0
C. botulinum type: A (Hall) B (green beans) C (ATCC 25766) D (ATCC 27517) E (Beluga) F 8G Pasteurized crabmeat Langeland
1
2
b
3 -
-
1.77 0.77 0.07 1.49 0.91 0.10 1.58 0.53 0.07
10,000 10,000 10,000
-
>2.0 >2.0 >2.0
10,000 10,000 200 2,000 10,000
0.13 0.66 0.06
C. sporogenes A1507 A1979
-
C. novyi (ATCC 17861)
-
C. perfringens (Lab)
0.10 0.07
Liver pate Tuna Crabmeat
1,000 100 0
NAb 1.11 0.08
A410 at a log1o dilution of: 1 2 3
>2.0 0.32
0.45 0.07
4
C
-
4
Log 1 0 dilution C. botulinum Type F toxin (ng/mI)
Culture/source or strains used
0.30
MLD, Mouse lethal dose. Highest 10-fold dilution at which two mice injected with toxin died. b NA, Not analyzed. c-, Less than or equal to 0.05.
Rabbit
2
1
Toxicity (MLD/ml)a
a
0._-
0
Product used
0 0
10 0
a MLD, Mouse lethal dose. Highest 10-fold dilution at which two mice injected with toxin died. b -, Less than or equal to 0.05.
commonly have low affinities for their homologous antigens. The double-sandwich ELISA detected type E C. botulinum toxin but only in undiluted culture fluids. In conclusion, the ELISA system using type F C. botulinum MAb can be used for rapid screening of suspected C. botulinum type Fcontaining samples. Compared with the 3-day mouse bioassay procedure, this double-sandwich ELISA system requires 1 day to assay and establish the toxicity of suspected culture supernatants or food samples. We thank D. Moore and C. Aulosio (Centers for Disease Control) for their assistance and C. Hatheway (Centers for Disease Control) for advice and clostridial cultures. In addition, we thank M. Hemphill, Food and Drug Administration, Atlanta, Ga., for technical assistance. LITERATURE CITED 1. Boroff, D. A., and G. Shu-Chen. 1973. Radioimmunoassay for type A toxin of Clostridium botulinum. Appl. Microbiol. 25: 545-549. 2. Ferreira, J. L., M. K. Hamdy, Z. L. Herd, S. G. McCay, and F. A. Zapatka. 1987. Monoclonal antibody for the detection of Clostridium botulinum type A toxin. Mol. Cell. Probes 1: 337-345. 3. Food and Drug Administration. 1984. Clostridium botulinum, p. 18.01-18.10. In Bacteriological analytical manual for foods, 6th ed. Association of Official Analytical Chemists, Arlington, Va. 4. Kamata, Y., S. Kozaki, T. Nagai, and G. Sakaguchi. 1985. Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin. FEMS Microbiol. Lett. 26: 305-309. 5. Kozaki, S., Y. Kamata, T. Nagai, J. Ogasawara, and G. Sakaguchi. 1986. The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin. Infect. Immun. 52:786-791. 6. Moller, V., and I. Scheibel. 1960. Preliminary report on the isolation of an apparent new type of C. botulinum. Acta Pathol. Microbiol. Scand. 48:80. 7. Notermans, S., A. M. Hagenaars, and S. Kozaki. 1982. The enzyme-linked immunosorbent assay (ELISA) for the detection and determination of Clostridium botulinum toxins A, B, and E. Methods Enzymol. 84:223-238. 8. Oguma, K., T. Agui, B. Syuto, K. Kimura, H. Iida, and S. Kubo. 1982. Four different monoclonal antibodies against type C, toxin of Clostridium botulinum. Infect. Immun. 38:14-20. 9. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulin-producing hybrid cell lines, p. 351-372. In B. B. Mishell and S. M. Shiigi (ed.), Selected methods in cellular immunology. W. H. Freeman and Company, San Francisco. 10. Sakaguchi, G. 1979. Botulism, p. 389-442. In H. Riemann and F. L. Bryan (ed.), Food borne infections and intoxications. Academic Press, Inc., New York. 11. Schantz, E. J., and D. A. Kautter. 1978. Standardized assay for Clostridium botulinum toxins. J. Assoc. Off. Anal. Chem.
61:96-99. 12. Shone, C., P. Wilton-Smith, N. Appleton, P. Hambleton, N. Modi, S. Gatley, and J. Melling. 1985. Monoclonal antibody-
VOL. 56, 1990
based immunoassay for type A Clostridium botulinuin toxin is comparable to the mouse bioassay. Appl. Environ. Microbiol. 50:63-67. 13. Tsuzuki, K., N. Yokosawa, B. Syuto, I. Ohishi, N. Jujii, K. Kimura, and K. Oguma. 1988. Establishment of a monoclonal antibody recognizing an antigenic site common to Clostridiiin botulinum type B, Cl, D, and E toxins and tetanus toxin. In-
NOTES
811
fect. Immun. 56:898-902. 14. Vermilyea, B. L., H. W. Walker, and J. C. Ayres. 1968. Detection of botulinal toxins by immunodiffusion. Appl. Microbiol. 16:21-24. 15. Yang, K., and H. Sugiyama. 1975. Purification and properties of Clostridiumn botuilinuin type F toxin. Appl. Microbiol. 29:598603.
p.
Vol. 56, No. 3
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0099-2240/90/030808-04$02.00/0 Copyright © 1990, American Society for Microbiology
NOTES
Monoclonal Antibody to Type F Clostridium botulinum Toxin JOSEPH L. FERREIRA,'* MOSTAFA K. HAMDY,2 STEVEN G. McCAY,l AND FRANCIS A. ZAPATKA' Food and Drug Administration, Atlanta, Georgia 30309,1 and Food Science Department, University of Georgia, Athens, Georgia 306022 Received 20 September 1989/Accepted 18 December 1989
Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was -10 mouse lethal doses of toxin per ml of toxic fluid.
culture fluids were assayed by an indirect ELISA system for antibody against type F toxin. Selected antibody-producing hybridoma cells were subcloned by limiting dilution in 96-well microdilution plates, and antibody-producing clones were then chosen. Antibody-producing hybridomas were expanded and used in Pristane (Sigma)-primed mice by i.p. injection of 106 hybridoma cells to produce ascites. An indirect ELISA system similar to that described by Ferreira et al. (2) was used to screen for antibody-producing hybridomas. Type F toxoid consisting of 0.5 jig of protein in 100 [l of carbonate buffer (0.1 M, pH 9.6) was placed in each well of the microdilution plate (Immulon II, round bottom; Dynatech Laboratories, Inc., Alexandria, Va.) and held overnight at 4°C. After three washings of each well with buffer A (PBS [pH 7.4] containing 0.1% bovine serum albumin [BSA], 0.05% Tween 20, and 0.02% sodium azide), nonspecific binding sites were blocked with 1% BSA in buffer A for 1 h at 35°C. Plates were then washed three times with buffer A, and 100 ,ul of hybridoma culture supernatants was placed in each well and incubated for 2 h at 35°C. Plates were again washed as described above, and 100 jl of alkaline phosphatase-labeled goat anti-mouse immunoglobulin G (IgG) conjugate (Sigma), diluted (1:1,000, vol/vol) in buffer A containing 1% BSA, was then added to each well. The plates were incubated at 35°C for 2 h and again washed four times as described above, and 100 [l of substrate (1 mg of p-nitrophenylphosphate per ml of glycine buffer [0.1 M, pH 10.4] containing 1 mM MgCl2 and ZnCl2) was added to each well. After 1 h of incubation at 35°C, the reaction was stopped by adding 25 jil of NaOH (3 M), and the A410 was measured spectrophotometrically. This basic assay was also used to examine normal mouse serum and ascites fluids by using type F toxin (WAKO Chemicals, Dallas, Tex.) and type F toxoid. A double-sandwich ELISA was used for the detection of type F toxin. In this procedure, ascites fluid from hybridoma 223 was diluted and dispensed into wells of a 96-well Dynatech microdilution plate. The plates were washed and then blocked by using 1% BSA. After the plates were washed, toxic culture supernatants, purified toxin preparations, or food samples were then added to each well. After sample incubation, plates were washed and rabbit polyclonal type F antitoxin (1.5 jig of protein per ml) was added to each
The first poisoning incident caused by type F Clostridium botulinum was observed in Denmark in 1958 (6). Type F toxin and other types of botulinum toxins can be detected by a variety of methods, including radioimmunoassay, gel diffusion, enzyme-linked immunosorbent assay (ELISA), and, currently the most sensitive procedure, the mouse bioassay (1, 7, 10, 11, 14). The lower sensitivities and specificities of the in vitro methods have been largely the result of the impurity of antigens used to produce the antibody (10). Monoclonal antibodies (MAbs) have been produced against toxin types A, B, C, D, and E of C. botulinum (2, 4, 5, 8, 12). In the present study, hybridomas were generated that produced MAbs against type F C. botulinum toxin. One MAb was used in combination with a double-sandwich ELISA system for the rapid detection of this toxin in foods, culture media, or other toxic preparations. BALB/c myeloma cell line Sp2/0-Ag-14NS(FO) was obtained from C. Aulosio, Centers for Disease Control, Atlanta, Ga., and was utilized for the production of type F hybridomas by fusion with spleen cells from an immunized BALB/c mouse. C. botulinum cultures (types A, B, C, D, E, and F), as well as cultures of other Clostridium spp. (C. sporogenes, C. novyi, and C. perfringens), were used to assess the specificity and sensitivity of the ELISA system. Type F C. botulinum toxin was isolated and purified by the method of Yang and Sugiyama (15). Female BALB/c mice were immunized as follows. Equal volumes of Freund complete adjuvant (Sigma Chemical Co., St. Louis, Mo.) were emulsified with F toxoid preparations in 0.06 M phosphatebuffered saline (PBS). The 0.5-ml emulsions, containing 100 jig of toxoid, were then injected intraperitoneally (i.p.) into mice. After 4 weeks, each mouse was again injected (i.p.) with 0.5 ml of toxoid (200 jug) in 0.06 M PBS. Three days after the second injection, the titers of the sera were measured and spleens were removed for fusion. Cells of the Sp2/0-Ag-14NS(FO) myeloma cell line used for fusion and type F hybridoma cells were grown in reinforced minimal essential medium (formula no. 78-5216; GIBCO Laboratories, Grand Island, N.Y.). The myeloma and spleen cells were fused at a 1:1 ratio by using 108 cells of each type following the procedure of Oi and Herzenberg (9). Tissue *
Corresponding author. 808
NOTES
VOL. 56, 1990
well. The plate was then washed, and alkaline phosphataselabeled goat anti-rabbit IgG conjugate (Sigma) was added. Again, after plates were washed, substrate solution (1 mg of p-nitrophenylphosphate per ml of glycine buffer [pH 10.4]) was added to each well. The reaction was then stopped, and the A410 was measured. The same procedure was also utilized with the rabbit polyclonal antibody serving as the first antibody and the hybridoma 223 MAb serving as the second antibody. The alkaline phosphatase-conjugated antimouse IgG and substrate were used as described above. To detect type F C. botulinum in foods, the Langeland strain was inoculated into oxygen-reduced food products (commercially canned liver pate, tuna fish, and crabmeat) and these products were incubated for 14 days at 32°C. Each product was then diluted (weight/volume) in PBS and assayed for the toxicity level (mouse lethal doses per milliliter) by using the mouse bioassay procedure of the Food and Drug Administration (3) and by the double-sandwich ELISA utilizing the type F MAb produced by hybridoma 223. Type F toxin from toxic culture fluid was also used to examine the neutralizing capacity of the MAb. A mixture of toxin and MAb was injected (i.p.) into Swiss mice, and the mice were observed for symptoms and death during 3 days of incubation. Isotyping was determined by an indirect ELISA. Antimouse sera (IgA, IgGl, IgG2a, IgG2b, IgG3, IgM [Sigma], kappa and lambda light chains [Miles Laboratories Inc., Elkhart, Ind.], IgD, and IgE [ICN Immuno-Biologicals, Lisle, Ill.]) were diluted 1:100 in 0.1 M carbonate buffer (pH 9.6) and refrigerated (4°C) overnight in Dynatech Immulon II microdilution plates. After plates were washed, mouse tissue culture fluids were incubated for 1 h at 24°C, plates were washed again, and alkaline phosphatase-labeled goat antimouse IgG (1:1,000 dilution) was added. After 1 h of incubation, the plates were washed and 1 mg of p-nitrophenylphosphate per ml of glycine buffer was added and incubated at 35°C for 30 min. The reaction was stopped by using 25 ,ul of 3 M NaOH per well, and the A410 was measured. Immuno-dot blots for toxin detection were prepared by diluting the WAKO toxin in PBS and 1 ,ul of toxin preparations and then were applied to nitrocellulose (NC) filters, allowed to dry for 10 min at 24°C, and blocked with PBS containing 0.3% Tween 20 (PBS-T) for 10 min. Ascites fluid, diluted 1:2,000 in PBS-T, was added to the NC and incubated for 1 h at 24°C. The NC was washed three times in PBS-T, and horseradish peroxidase-conjugated anti-mouse IgG (Sigma) (diluted 1:100 in PBS-T) was incubated with the NC for 1 h at 35°C. The NC was washed three times in PBS-T and then once with PBS before the substrate (0.5 mg of 3,3'-diaminobenzidine per ml of 0.003% H202 [Sigma]) was added. The substrate was incubated for 15 min, and the reaction was stopped by using fresh distilled water. MAb-producing hybridomas were generated against type F C. botulinum toxin. The titer of hybridoma 223 in ascites was assayed by the indirect ELISA system by using type F botulinum toxin (Fig. 1), and this analysis revealed a titer of _106/ml. Approximately 33% of the stable hybridomas produced IgM; the remainder synthesized IgGl or IgG2a. Figure 2 shows the double-sandwich ELISA results of various MAb dilutions (ascites of hybridoma 223), polyclonal type F mouse serum, and normal mouse serum against 0.5 ,ug of type F toxin per well. It is evident that the hybridoma 223 ascites titer is -100 times higher than that of polyclonal type F serum. The sensitivity of the doublesandwich ELISA system for the detection of type F toxin as a function of dilution (nanograms per milliliter) was observed to increase when the MAb rather than the polyclonal rabbit
809
0.4-
---
0.4--b
E 0 V-
0.3-
r_
S 0.2-\
0 .0 c .0 D0
.0
0.1
-
0.0
-
0 0
2
4 3
2
I
6 5
4
6
7
Log10 dilution of ascites fluids FIG. 1. Indirect ELISA assay of hybridoma 223 ascites fluid against 0.1 jig of C. botulinum type F toxin per well. Tenfold dilutions were tested and showed a titer of -106/ml.
serum was used as the first antibody (Fig. 3). No crossreactions were observed with other toxins of C. botulinum types A, B, or C, but the MAb did react slightly with toxin of C. botulinum type D. No reaction was observed with nontoxic C. novyi, C. perfringens, or C. sporogenes strains. However, type E reacted strongly when only undiluted culture supernatants were used (Table 1). When toxic foods were examined in the double-sandwich ELISA, toxicity was determined at levels equivalent to 10 mouse lethal doses per ml (Table 2). The immuno-dot blot examination of WAKO toxin was positive at a dilution of 5 ,ug/ml or a total of 5 ng of toxin on NC filters. The isotype of the hybridoma 223 MAb was determined to be IgG2a heavy chain and kappa light chain by the ELISA. Antibody produced by hybridoma 223 was chosen on the basis of its high titer and specificity for type F C. botulinum. We found that when the MAb was attached to the plate first this system gave added sensitivity over the rabbit serum (polyclonal) being used as the first antibody. This assay was also equally sensitive for detecting C. botulinum type F in culture fluids, foods, and toxic preparations made from type F toxin. Of 14 hybridomas selected from fusion plates for
1.6E o
1.2-
4
0.8
223 Ascites
0 0 .0
o
o
C U .0
Poly F
0.4
0.4-
0
Normal
0.0-1 I I 3
i
Log10 dilution
5
mouse
6
antibody
FIG. 2. Double-sandwich ELISA of mouse sera (normal and F polyclonal) and hybridoma 223 ascites fluid against 0.5 ,ug of type F Wako toxin per well. Tenfold dilutions were tested. The hybridoma 223 ascites titer is 100 fold greater than than of F polyclonal serum.
APPL. ENVIRON. MICROBIOL.
NOTES
810
TABLE 2. Detection of type F toxin in foods by using the double-sandwich ELISA and MAb 223
E
2
Mob 223
0 _E
°
0
c
aw C
°0
0
1
0
.0
.0
0
0 / 0-
O-
3
FIG. 3. Effect of first antibody (MAb from hybridoma 223 or rabbit polyclonal serum) on detection of type F Wako toxin in double-sandwich ELISA. The sensitivity increases when MAb rather than polyclonal rabbit serum is used as the first antibody.
expansion, only one (designated as hybridoma 223) was used in the double-sandwich ELISA system for toxin detection. This hybridoma was selected for its high titer and specificity for type F toxin. The MAb produced by hybridoma 223 used in the double-sandwich ELISA method did not neutralize type F toxin in 20-g mice. In the toxin molecule, there may be more than one epitope present and, since the MAb can react with one specific antigenic site or conformation, it is possible that other epitope(s) would be available to cause botulism in the mouse. It is conceivable that the MAb-toxin complex may dissociate in vivo following its injection. If the MAb has a low affinity for a particular toxin epitope, under certain conditions it would not protect the animal in toxin neutralization testing. Another possible explanation is that in similar but not identical antigenic determinants (epitopes) binding of the MAb occurs but with low affinity. MAbs TABLE 1. Cross-reaction of MAb 223 to different types and strains of C. botulinum and other clostridia by using double-sandwich ELISA Absorbance at a log1o dilution of:
Toxicity
(MLD/ml)a 0
C. botulinum type: A (Hall) B (green beans) C (ATCC 25766) D (ATCC 27517) E (Beluga) F 8G Pasteurized crabmeat Langeland
1
2
b
3 -
-
1.77 0.77 0.07 1.49 0.91 0.10 1.58 0.53 0.07
10,000 10,000 10,000
-
>2.0 >2.0 >2.0
10,000 10,000 200 2,000 10,000
0.13 0.66 0.06
C. sporogenes A1507 A1979
-
C. novyi (ATCC 17861)
-
C. perfringens (Lab)
0.10 0.07
Liver pate Tuna Crabmeat
1,000 100 0
NAb 1.11 0.08
A410 at a log1o dilution of: 1 2 3
>2.0 0.32
0.45 0.07
4
C
-
4
Log 1 0 dilution C. botulinum Type F toxin (ng/mI)
Culture/source or strains used
0.30
MLD, Mouse lethal dose. Highest 10-fold dilution at which two mice injected with toxin died. b NA, Not analyzed. c-, Less than or equal to 0.05.
Rabbit
2
1
Toxicity (MLD/ml)a
a
0._-
0
Product used
0 0
10 0
a MLD, Mouse lethal dose. Highest 10-fold dilution at which two mice injected with toxin died. b -, Less than or equal to 0.05.
commonly have low affinities for their homologous antigens. The double-sandwich ELISA detected type E C. botulinum toxin but only in undiluted culture fluids. In conclusion, the ELISA system using type F C. botulinum MAb can be used for rapid screening of suspected C. botulinum type Fcontaining samples. Compared with the 3-day mouse bioassay procedure, this double-sandwich ELISA system requires 1 day to assay and establish the toxicity of suspected culture supernatants or food samples. We thank D. Moore and C. Aulosio (Centers for Disease Control) for their assistance and C. Hatheway (Centers for Disease Control) for advice and clostridial cultures. In addition, we thank M. Hemphill, Food and Drug Administration, Atlanta, Ga., for technical assistance. LITERATURE CITED 1. Boroff, D. A., and G. Shu-Chen. 1973. Radioimmunoassay for type A toxin of Clostridium botulinum. Appl. Microbiol. 25: 545-549. 2. Ferreira, J. L., M. K. Hamdy, Z. L. Herd, S. G. McCay, and F. A. Zapatka. 1987. Monoclonal antibody for the detection of Clostridium botulinum type A toxin. Mol. Cell. Probes 1: 337-345. 3. Food and Drug Administration. 1984. Clostridium botulinum, p. 18.01-18.10. In Bacteriological analytical manual for foods, 6th ed. Association of Official Analytical Chemists, Arlington, Va. 4. Kamata, Y., S. Kozaki, T. Nagai, and G. Sakaguchi. 1985. Production of monoclonal antibodies against Clostridium botulinum type E derivative toxin. FEMS Microbiol. Lett. 26: 305-309. 5. Kozaki, S., Y. Kamata, T. Nagai, J. Ogasawara, and G. Sakaguchi. 1986. The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin. Infect. Immun. 52:786-791. 6. Moller, V., and I. Scheibel. 1960. Preliminary report on the isolation of an apparent new type of C. botulinum. Acta Pathol. Microbiol. Scand. 48:80. 7. Notermans, S., A. M. Hagenaars, and S. Kozaki. 1982. The enzyme-linked immunosorbent assay (ELISA) for the detection and determination of Clostridium botulinum toxins A, B, and E. Methods Enzymol. 84:223-238. 8. Oguma, K., T. Agui, B. Syuto, K. Kimura, H. Iida, and S. Kubo. 1982. Four different monoclonal antibodies against type C, toxin of Clostridium botulinum. Infect. Immun. 38:14-20. 9. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulin-producing hybrid cell lines, p. 351-372. In B. B. Mishell and S. M. Shiigi (ed.), Selected methods in cellular immunology. W. H. Freeman and Company, San Francisco. 10. Sakaguchi, G. 1979. Botulism, p. 389-442. In H. Riemann and F. L. Bryan (ed.), Food borne infections and intoxications. Academic Press, Inc., New York. 11. Schantz, E. J., and D. A. Kautter. 1978. Standardized assay for Clostridium botulinum toxins. J. Assoc. Off. Anal. Chem.
61:96-99. 12. Shone, C., P. Wilton-Smith, N. Appleton, P. Hambleton, N. Modi, S. Gatley, and J. Melling. 1985. Monoclonal antibody-
VOL. 56, 1990
based immunoassay for type A Clostridium botulinuin toxin is comparable to the mouse bioassay. Appl. Environ. Microbiol. 50:63-67. 13. Tsuzuki, K., N. Yokosawa, B. Syuto, I. Ohishi, N. Jujii, K. Kimura, and K. Oguma. 1988. Establishment of a monoclonal antibody recognizing an antigenic site common to Clostridiiin botulinum type B, Cl, D, and E toxins and tetanus toxin. In-
NOTES
811
fect. Immun. 56:898-902. 14. Vermilyea, B. L., H. W. Walker, and J. C. Ayres. 1968. Detection of botulinal toxins by immunodiffusion. Appl. Microbiol. 16:21-24. 15. Yang, K., and H. Sugiyama. 1975. Purification and properties of Clostridiumn botuilinuin type F toxin. Appl. Microbiol. 29:598603.
