detection of clostridium botulinum type e toxin by monoclonal antibody enzyme immunoassay

16
DETECTION OF CLOSTRZDZUM BOTULZNUM TYPE E TOXIN BY MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY PHILIP C.K. WONGl U. S. Food and Drug Administration Los Angeles, California 90015 Accepted for Publication January 15, 1996 ABSTRACT Botulinum type E toxin is a well recognized causative agent of seafood botulism poisoning. Underprocessingor postretort recontamination of preserved seafoods has resulted in sporadic cases of botulism. Currently, laboratory mice are being used to detect this toxin. However, it requires three to six days to obtain final results. A rapid method using monoclonal antibody (Mab) enzyme immunoassay was therefore developed. Hybridomas secreting spec@ Mab against the type E epitope were generated by fusion of SP/2O-Ag14 myeloma cells with spleen cells from BALBIc mice immunized with botulinum type E neurotoxoid. Five potent, stable hybridomas were selected, cloned, propagated, and preserved in liquid nitrogen as cell lines. Immunoglobulin subisotyping showed these Mabs belong- ed to the IgG subclasses. No cross-reaction was observed with culture supernatants of C. botulinum types A , B, and F or with crude toxins extracts of type C and D. Large quantities of Mabs were produced in ascitesfluids, harvested, and af- Jinitypunfled. A Mab-based biotin-avidinamplijied double sandwich enzyme-linked immunosorbent assay allowed detection of type E toxin in inoculated seafoods at levels equivalent to 1-10 MLDs/ml (5-10 pg/ml). INTRODUCTION Clostridium botulinum is an anaerobic, sporeforming, foodborne, bacterial pathogen which elaborates a potent neurotoxin which causes high mortality and morbidity rates. There are currently seven known types of C. botulinum toxin lCorresponding author: U.S. Food and Drug Administration (HFR-PA260), 1521 W. Pic0 Boulevard, Los Angeles, CA. 90015. Phone: (213:) 252-7575. Fax: (213) 251-7142. Journal of Rapid Methods and Automation in Microbiology 4 (1996) 191-206. All Rights Reserved. 0 Copyright 1996 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 191

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DETECTION OF CLOSTRZDZUM BOTULZNUM TYPE E TOXIN BY MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

PHILIP C.K. WONGl

U. S. Food and Drug Administration Los Angeles, California 90015

Accepted for Publication January 15, 1996

ABSTRACT

Botulinum type E toxin is a well recognized causative agent of seafood botulism poisoning. Underprocessing or postretort recontamination of preserved seafoods has resulted in sporadic cases of botulism. Currently, laboratory mice are being used to detect this toxin. However, it requires three to six days to obtain final results. A rapid method using monoclonal antibody (Mab) enzyme immunoassay was therefore developed. Hybridomas secreting spec@ Mab against the type E epitope were generated by fusion of SP/2O-Ag14 myeloma cells with spleen cells from BALBIc mice immunized with botulinum type E neurotoxoid. Five potent, stable hybridomas were selected, cloned, propagated, and preserved in liquid nitrogen as cell lines. Immunoglobulin subisotyping showed these Mabs belong- ed to the IgG subclasses. No cross-reaction was observed with culture supernatants of C. botulinum types A, B, and F or with crude toxins extracts of type C and D. Large quantities of Mabs were produced in ascites fluids, harvested, and af- Jinity punfled. A Mab-based biotin-avidin amplijied double sandwich enzyme-linked immunosorbent assay allowed detection of type E toxin in inoculated seafoods at levels equivalent to 1-10 MLDs/ml (5-10 pg/ml).

INTRODUCTION

Clostridium botulinum is an anaerobic, sporeforming, foodborne, bacterial pathogen which elaborates a potent neurotoxin which causes high mortality and morbidity rates. There are currently seven known types of C. botulinum toxin

lCorresponding author: U.S. Food and Drug Administration (HFR-PA260), 1521 W. Pic0 Boulevard, Los Angeles, CA. 90015. Phone: (213:) 252-7575. Fax: (213) 251-7142.

Journal of Rapid Methods and Automation in Microbiology 4 (1996) 191-206. All Rights Reserved. 0 Copyright 1996 by Food & Nutrition Press, Inc., Trumbull, Connecticut. 191

192 P.C.K. WONG

(A, B, C, D, E, F, and G) which differ from each other by their antigenic specifici- ty, relative toxicity, host specificity and food sources of intoxication.

The structure of C. botulinum toxin consists of two components: a nontoxic protein subunit (M.W. approximately 500,000), and the neurotoxin (M.W. a p proximately 150,000). The bimolecular complex of nontoxic protein subunit and neurotoxin is referred to as “progenitor toxin,” and the isolated neurotoxin as “derivative toxin. ” The neurotoxin, formed in the bacterium, is excreted either as a single chain (native, nonactivated posttranslational protein with low toxici- ty) as in type E or as in proteolytic types A, B, and F as di-chain (fully activated molecule by a protease produced by the bacterium to a highly toxic form) com- posed of a heavy (M.W. approx. lOO,OOO), and a light chain (M.W. approx. 50,000) linked by disulfide bond@). Trypsin treatment converts the type E neurotoxin into the diichain form having maximal potential toxicity. (DasGupta and Rasmussen 1984; Kosaki et al. 1986; Sathyamoorthy and DasGupta 1985; Smith and Sugiyama 1988; Whelan et al. 1992). Both the light and the heavy chains act synergistically to inhibit acetylcholine release from the nerve endings resulting in neuromuscular paralysis (Sathyamoorthy and DasGupta 1985; Smith and Sugiyama 1988; Whelan ef al. 1992).

Type E botulism is primarily associated with seafood because the ecological niches occupied by this Clostridium botulinum type E strains are primarily aquatic (Badhey et al. 1986; Smith and Sugiyama 1988). Underprocessing or postretort recontamination of seafoods preserved by smoking or canning has resulted in sporadic outbreaks of botulism caused by type E toxin (Badhey et al. 1986; Gib- son ef al. 1988 and Telzak er al. 1990). The FDA laboratory test procedures which are conducted to detect and identify botulinum toxin(s) in foods (i.e., the mouse toxicity and protection/typing technique) are prescribed by the official A.O.A.C./FDA Bacteriological Analytical Manual, (Kautter et al. 1992). This is the most sensitive laboratory test known, since it is able to detect as low as two minimum lethal doses (MLD/ml) toxin by intraperitoneal (i.p.) injection of mice with food extracts or culture supernatants (Kautter et al. 1992). However, the test requires 3 to 6 days to obtain final results. The need for monitoring the mice for deaths with typical symptoms of botulism is a mandatory requirement and subsequent mouse protection test series have to be performed for specific detection of preformed and/or cultural toxin type(s) in the implicated food(s).

Laboratory methods, other than the mouse bioassay, have been developed for botulinum toxin detection. These include the cytotoxicity of animal cell lines, immunodiffusion, hemagglutination, radio-immunoassay (RIA), and enzyme- linked immunosorbent assay (ELISA). However, except for the RIA, all of these methods lack the sensitivity and simplicity of the conventional mouse bioassay. Ferreira et al. (1987), Gibson et al. (1987), Modi et al. (1986) and Notermans et al. (1984) described the use of monoclonal antibody-based ELISA procedures

DETECTION OF C. BOTULJNUM TYPE E TOXIN 193

for the detection of C. botulinum type A, or type B, and types A and B toxins with sensitivities comparable to that of the mouse bioassay. More recently, Pot- ter et al. (1993) used polyclonal antibodies based-ELISA for the detection of botulinum toxins type A, B, and E in inoculated food samples. False positives generated by the reaction with the hemagglutinin subunit of the toxin were en- countered. Doellgast et d. (1994:) described the use of enzyme-linked coagulase with dual-label antibodies for the detection of C. botulinum neurotoxins A, B, and E. However, the test was extremely costly.

The purpose of this research was designed to apply state-of-the-art hybridoma technology to generate Mabs specific for type E toxin and to investigate the use of Mab-based avidin-biotin amplification (Shamuddin and Harris 1983) double sandwich ELdSA system for a rapid, sensitive, specific, and cost-effective method for the detection of preformed type E toxin in simulated commercial canned seafoods and toxin in media broths.

MATERIALS AND METHODS

Toxin and Toxoid

Purified type E neurotoxin was procured from R.B. DasGupta of the Universi- ty of Wisconsin, Madison, Wis. The toxin was converted to toxoid with 0.4% formaldehyde by the method described in Ferreira et al. (1987) modified by ter- minating detoxification in 7 days. A sample of the toxoid was tested for complete detoxification by the mouse bioassay. Also, a toxoid sample was subjected to SDS-PAGE using precast gradient gels and known prestained molecular weight standards (Integrated Separation System, Hyde Park, MassJ along with a toxin sample. Protein migration bands were directly measured on the gel using Elec- trophoresis Rf Reader (Sigma, product No. S 2010).

Immunization, Cell Culture, and Fusion

BALB/c mice were immunized intraperitoneally with primary and secondary doses of toxoid (30 pg each) in 0.2ml Ribi adjuvant (RIB1 Immunochem Research, Inc., Hamilton, Mont.) following the manufacturer’s instructions. The mice were tail-bled to determine their serum antibody titers by both the Ouchterlony plate and the ELISA techniques. A final booster dose (50 pg) in 0.2 saline was ad- ministered, intravenously, without adjuvant three days prior to cell fusion. A nonsecreting mouse myeloma frozen cell line stock, SFWO-AG14, was procured from the repository of the American Type Culture Collection (ATCC, CRL 1581, Rockville, Md). It was subcultured in a growth medium consisting of DMEM

194 P.C.K. WONG

supplemented with 10% fetal bovine serum, 1% 20 mM/ml L-glutamine, 2% penicillin (5000 units)-streptomycin (5 mg) solution (Sigma Chemicals, St. Louis, MO) and incubated at 37C in 5% C02 cell culture incubator. Cell stocks were cryogenically preserved in a freezing medium. To ensure the myeloma cells were free from revertants, a sample of the frozen cells was subcultured in the presence of 8-azaguanine (Sigma) prior to cell fusion. Fusion cell partners were mixed at a ratio of 1 x lo7 log phase myeloma cells to 5 x lo7 spleen cells following the Boehringer-Mannheim PEG 1500 Product Application Scheme in which company-prepared polyethylene glycol (MW 1500 f 100) was used as the fusogen. After centrifugation and removal of the fusogen, the fusion products were resuspended in 100 ml HAT selection medium (growth medium supplemented with 1 x HAT concentrate (Sigma), 5 pg/ml STM (RIBI), and 10% Hybridoma Cloning Factor (HCF) (Igen Inc., Rockville, Md.) in place of a feeder cell layer. Aliquots of 200 p1 were added to well of 96-well cell culture plates (Costar). Ten wells containing nonPEG treated cell mixture in HAT selection medium were included as controls. The plates were then incubated as before. The post fusion cells were observed daily for the outgrowth of hybridomas.

Hybridoma Screening, Mab Specificity Testing and Immunoglobulin Subisotyping

Supernatants from all the wells showing hybridoma outgrowths were screened for monoclonal antibodies (Mab) recognizing the trypsinated type E toxin an- tigen by the Mouse IgG Monoclonal Antibody ELISA Screening Kit (Hyclone Laboratories, Inc., Logan, Utah) following the manufacturer’s supplied ELISA procedure. Briefly, the Mabs to be screened were allowed to react with the im- mobilized trypsinated type E toxin antigen (3 pg/ml) in the first step and in the second step the amount of Mab bound to the antigen was measured using an en- zyme labelled second antibody. Parallel study was conducted to determine if randomly chosen clonal fluids from the screened positive hybrid clones also recognized the nontrypsinated type E toxin by the same ELISA method. Clonal supernatants from selected hybridomas were tested for cross-reaction with crude extracts of botulinum toxin types A, B, C, D, and F (Wako Bioproducts, Rich- mond, Va.) coated at 5 pg/ml, again, using the ELISA as previously. Mabs from specific producing hybridomas were subisotyped as to their immunoglobulin subclasses (IgG1, IgG2a, IgGZb, IgG3, IgA, and IgM) using the procedure out- lined in the Mouse Mab ELISA Subisotyping kit and Mab subisotyping Control Proteins (Hyclone).

DETECTION OF C. BOTUHNUM TYPE E TOXIN 195

Cloning, Ascites Production, and Purification

Selected hybridomas were expanded, and again tested for secreting specific Mab using the ELISA as before. Nonstable, short-lived, and poor secreters were eliminated from further propagation. Stable, high-titer producing hybridomas were cloned twice by the limiting dilution technique to purify the clones and to ensure their monoclonality. Cloned hybridomas were cryogenically preserved as cell lines. For ascites production, approximately 1.0 x lo6 log phase hybridoma cells in physiological saline was injected into the peritoneal cavity of each mouse primed with pristine (Sigma). The ascites fluids were collected after about 10 days. Pooled centrifuged and defatted ascites fluids were purified using the protein G affinity column (G) IgG Purification Kit (Pierce, Rockford, Ill.) according to the technical supplied information. The eluted IgG fractions collected were assayed for anti- body concentration at 280 nm using a UV/VIS Lambda 3 spectrophotometer (Perkin Elmer, Foster City, Ca.), and the fractions containing the IgG were stored frozen at - 20C.

Detection of Type E Toxin in Foods and in Culture Supernatants

a. Spiked Foods. Commercial sterile (heat treatment in canning designed to destroy viable mesophilic vegetative microorganisms and spores of public health significance) canned baby clams, salmon, and oysters were purchased from local supermarkets. Portions of 50-100 g were aseptically removed from each of the above food items and placed in 15 x 2 cm screw-cap tubes. Sterile distilled water (20-25 ml) was added to dilute the sodium/potassium salts and other inhibitory/an- tibotulinal agents that might be present in the products, and the tubes were retorted to simulate canning process for commercial sterility. After cooling, an inoculum of approximately 1 x lo3 CFU/O.l ml of a type A, B, E, F C. botulinum strain was seeded into each of the tubes, and incubated at 35C or 25C (type E) for 7 to 10 days.

b. Cultural Toxins. Proteolytic C. botulinum types A, B, C, D as well as other clostridial species were grown in cooked meat broth at 35C for 7 days. C. botulinum type E, and proteolytic type F strains were grown in trypticase-peptone- glucose-yeast-extract (TPGY) broth at 25C and 35C respectively, for 7 to 10 days. The cultural strains and their sources are shown in Table 1.

c. Determination of Mouse Minimal Lethal Dose (MLD/ml). At the end of incubation, toxic food supernatants and culture broths were centrifuged in metal tubes to pellet cells and debris. The type E toxin was trypsinated according to

196 P.C.K. WONG

TABLE 1, TEST STRAINS AND TOXINS

Organisms StrainsfToxins Source'

C. botulinum type A 6-6-85 A

11 17843 B B It C Wako C toxin C I D Wako D toxin C

It E 6-0-85 A

It E Beluga

II F Lange 1 and

n E 9564

n G 33253

C. sporogenes B/F C. perfringens PBF-1 C . tetani 10779 c. butyricum 8260 C . novyi 19402 B '. A=FDA culture collections; B=American type culture

collections; C=Wako Co.

Kautter ef al. (1992). The trypsinated type E toxin and other proteolytic botulinum type toxin supernatants were then tested for their toxicity and toxin titer (MLD/ml) which was determined from the highest 10-fold serial dilution of the toxic super- natant injected (i.p.) into pair of mice (0.5 ml/mouse) killing both in 3 days ac- cording to the mouse toxicity test (Kautter el al. 1992).

d. Treatment of Food Supernatants and Cultural Broths for Double- Sandwich ELISA. Aliquots of 3.5 ml of each supernatant from c. above were centrifuged for desalting and removal of unwanted membrane-pemieating pro- teins and peptides species from nonpermeating toxin species by ultrafiltration with the 50K Filtron Microsep device (Filtron Technology Corporation, Northborough, MA). The final volume was approximately 0.5 ml to 1.0 ml which was restored to 3.5 ml with 0.05M phosphate buffer, pH 6.0. This process of concentra- tionlredilution was repeated 2 x or more to maximize microsolute clearance and background reduction.

e. Amplified Double-Sandwich ELISA for Type E Toxin Detection. The principle underlining the development of this amplification technique was based on the high affinity and multiple binding capability of avidin for biotin. The analytical protocol, after much optimization, followed was the Pierce Immunohre

DETECTION OF C. BOTULINUM TYPE E TOXIN 197

Ultra-Sensitive ABC Peroxidase Rabbit IgG Staining Kit (Pierce) in which the company-prepared ultra reagents Avidin and Biotinylated peroxidase were used as the amplifier. Briefly, wells of a microplate (Immulon 4, Dynatech) was coated with 100 pl aliquots of a solution containing approximately 10.0 pg/ml of Mab to type E toxin (Clone #4E7). After overnight incubation at 4C and washings with PBS/tween, the plate was blocked with SuperBlock blocking buffer (Pierce) at 25C for 30 rnin and washed as previously. Wells (in duplicates) were loaded with 100 pl aliquots of type E-containing supernatants of culture media or food extract obtained from d. above. Both undiluted and, depending on their toxin content (MLDs/ml) 10-fold serial diluted samples in phosphate buffer pH 6.0 were being used. Other wells (also in duplicates) were loaded with 100 p1 ali- quots of undiluted culture and food supernatants from other types of C. botulinum, or with undiluted culture supernatants of other clostridial species, and reagent controls. The plate was incubated for 1 h at 25C and then washed 5 x . Thereafter, 100 p1 aliquots of diluted rabbit anti-E affinity purified polyclonal antibodies (NCTR, Jefferson, AR) was added. After incubation for 1 h at 25C the plate was washed 6 x . To each well 100 pl of biotinylated goat anti-rabbit IGg (Gibco) was added and incubated for 1 h at 25C. After 30 min the ultra-se nsitive avidin- biotinylated peroxidase premix (Pierce) was prepared and incubated for 30 min to allow the complex to form. The plate was then washed 6x and 100 pl/well of the complex was added followed by incubation for 30 min at 25C and the plate was washed as before. Finally, 100 pl/well aliquots of o-phenylenediamine (OPD) substrate solution was added and incubated at 25C for 5-10 min. The enzyme reaction was stopped by adding stop solution. The reaction product (brownish orange color) was measured at 492 nm using a microplate reader (SLT Labinstruments) which was connected to a printer. The threshold positive O.D. value was statistically determined from the mean of all negative controls plus 3 standard deviations of the mean. The experiment was repeated 2 times for reproducible results.

RESULTS

Properties of the Toxin and Toxoid

The molecular structure of the botulinum type E derivative toxin consists of one chain whereas, the progenitor (crude) toxin consists of two subunits as revealed by single and double bands on SDS-PAGE gel. (Fig. 1, lanes: 5 ,6 and 3,8, respec- tively). By comparison, the toxoid run on the same gel appeared to be diffuse and heterogeneous. It consisted of fractions having molecular weights higher and lower than 150,000 (visible and measurable on gel; not illustration documented) with an average M.W. of 175,OoO(Fig. l,lanes:4,7, and Table 2). These changes

198 P.C.K. WONG

FIG. 1. SDS-PAGE OF C. BOTULINUM TYPE E TOXIN AND TOXOID Lanes 1, 10: mid-range protein molecular weight markers. 2, 9: myosin marker protein. 3, 8: crude (progenitor) C. borulinurn type E toxin preparation (Wako). 5 , 6 : purified C. botulinurn type E derivative toxin purchased from U. of Wiscon- sin. 4, 7: C. borulinurn type E toxoid preparation. (Diffuse band seen on gel, not

illustration documented).

have been shown to result from polymerization/aggregation effects caused by for- maldehyde in the detoxification process (DasGupta and Rasmussen 1984). The toxicity of the protein was destroyed without altering its antigenicity. This was shown by the Ouchterlony immunodiffusion technique wherein antibody present in the mouse serum formed precipitin lines of equal sharpness and intensity with either the toxoid or toxin loaded in the center well (Fig. 2 and 3).

Establishment of Hybridomas and Some Properties of the Mabs Against the Type E Toxin

Of the 480 wells plated 150 showed hybrid outgrowths, giving a fusion effi- ciency of 3 1 % . Of the 150 hybrids, about 37 produced antibody against the tryp- sinated type E toxin (Fig. 4). Parallel study indicated that positive clonal super- natants recognized the type E toxin in both the trypsinated and the nontrypsinated forms (Table 3). Finally, after clonings and selection, five stable, specific, and high-titer producing hybridoma clones (1B9, 3C2, 4C5, 4F3, and 4E7) were established as hybridoma cell lines and preserved in liquid nitrogen. The Mabs

DETECTION OF C. BOTULINUM TYPE E TOXIN 199

TABLE 2.

BY SDS-PAGE DETERMINATION OF PROTEIN TOXIN MOLECULAR WEIGHT

Protein Molecular Weight Migration standardsttoxin

(daltons) Distance (mm)

Myosin

Phosphorylase B

Glutamate dehydrogenasa

Ovalbumin

Carbonic anhydrase

Lactoglobulin

Cytochrome C

C. botulinum type E toxin (U. Wisconsin)

Crude C. botulinum E toxin (Wako) Type E toxoid (visible and readable on gel)

200,000

95,000

55,000

43,000

29,000

18,400

12,000

150,000

150,000 and 125,000

175,000 (average)

15

30

39

43

50

53

50

21

21, 24

19 to 22

FIG. 2. OUCHTERLONY IMMUNODIFFUSION TECHNIQUES IN WHICH THE ANTIGEN EMPLOYED WAS TYPE E TOXOID

Center well charged with toxoid. Peripheral wells charged with sera from immunized mice. Precipitin lines joined together indicated reaction of identity.

200 P.C.K. WONG

FIG. 3. OUCHTERLONY IMMUNODIFFUSION TECHNIQUE IN WHICH THE ANTIGEN EMPLOYED WAS TYPE E TOXIN

Center well was charged with toxin. Peripheral wells were charged with doubling dilution of mouse antiserum. The well in the 3 o’clock position contained a normal

mouse serum.

FIG. 4. ELISA PLATE ASSAY OF HYBRIDOMA CLONES The appearance of a brownish-yellow color in the wells is indicative of a positive

test as compared to negative and reagent control blanks.

produced on a large scale in ascitic fluids were harvested, purified, and stored at -2OC. Immunoglobulin subisotyping of the Mabs revealed the antibodies to be of the IgGl, IgG2a, and IgG2b subclasses (Table 4). No cross-reaction was

DETECTION OF C. BOTULINUM TYPE E TOXIN 20 1

exhibited with crude type A, B, C, D and F toxin extracts by the ELISA test (Table 5).

TABLE 3.

DETERMINING CLONAL FLUIDS FROM SELECTED POSITIVE HYBRID CLONES REACTING WITH BOTH NON-TRYPSINATED AND

TRYPSINATED TYPE E TOXIN BY ELISA

Hybrid clonal O.D. reading at 492 m fluids

Non-Trypsinated' Trypsinated' -

B1D7 0.283 0.239

B3C2 0.385 0.428

B3D4 0.353 0.392

B4D3 0.142 0.148

B4C5 0.430 0.440

C1B9 0.399 0.400

A4E7 0.282 0.287

F1D5 0.406 0.459

F1E5 0.263 0.305

F1B7 0.231 0.245

0.052

0.059

0.055

0.057

a.Wells coated at 5.3 ug/ml/well

b.Medium control

c.Conjugate control

Detection of Type E Toxin in Culture Supernatants and in Foods Using the avidin-biotin amplified ELISA, trypsinated type E toxin in culture

supernatants and in food extracts, in duplicate runs, can be detected at a level equivalent to 1-10 MLD/ml (Table 6). The positive O.D. threshold value was 0.157 when the mean of all negative control vahes was 0.085 and the standard deviation 0.024. No cross-reaction was observed with the culture supernatants of C. botulinum type A, B, F G , C. sporogenes, C. tetani, C. butyricum, C. novyi, and C. pe@ingens.

202 P.C.K. WONG

TABLE 4. SUBISOTYPING OF MONOCLONAL ANTIBODIES BY ELISA

~~ ~

Mab clonal supernatants'

lBgb 4C5 4F3 4 E7 3C2

0.084 0.053 0.053 0.058 0.125

0.111 0.100 0.108 0.058 0.067

0.047 0.050 0.053 0.184 0.056

0.052 0.055 0.062 0.061 0.058

~~

Subisotyping control proteins'

0.154 IgGl

0.126 IgGZa

0.252 IgG2b

0.238 IgG3

0.051 0.051 0.059 0.062 0.069 0.195 IgGA

0.051 0.051 0.052 0.051 0.069 0.210 IgGM

0.047 0.047 0.046 0.045 0.048 0.050 l%BSA

'Numbers represent ELISA values at 492m. blB9 is an IgGZa, 4C5 is an IgGZa, 4F3 is an IgGZa, 4E7 is an IgGZb, 3C2 is an IgG1.

TABLE 5. MAEl SPECIFICITY DETERMINATION AGAINST OTHER TYPES OF

CRUDE BOTWLINAL TOXINS BY ELISA

Botulinal

Toxin Types' Mabs ELISA O.D. at 492 m

1B9 3C2 4F3 4c5 4 E7

0.070 0.074 0.072 0.081 0.076 0.071 0.072 0.073 0.082 0.075 0.073 0.076 0.075 0.005 0.080 0.076 0.075 0.073 0.001 0.070 0.220 0.215 0.216 0.201 0.232 0.000 0.082 0.086 0.007 0.079

a.Crude toxin (Wako) coated at 5 Mg/ml/well

DISCUSSION

ELISA determinations depend on the target antigen specificity of the im- munoglobulin in order to recognize and quantify low levels of specific protein. This was accomplished by the use of highly purified type E neurotoxin for its

DETECTION OF C. BOTULINUM TYPE E TOXIN 203

TABLE6 DETECTION OF TYPE E TOXIN IN CULTURAL BROTHS AND EXTRACTS OF

CANNED SEAFOOD PRODUCTS INOCULATED WITH VARIOUS CLOSTRIDIA USING MAE BASED-AVIDIN-BIOTIN AMPLIFIED ELISA

Test Toxin' ELISA optiul Density (0.D.p u 492 m Organism/Food Titer (bp 10 dilulion)

MLDlml 0 -1 -2 -3 4 -5 floe 10)

c. botulinum toxin T y p e A 0 7 D A M ) 5 0.078

Type c OHM0 CO) 6 0.068 Type D WAUO CO) 6 0.066

Type B (ATCC 17843) 5 0.075

Type B (ATCC 9564) 4 1.641 0.926 0.373 0.138 0.123 TypcE.WA6-0-W 4 1.331 0.869 0.371 0.282 0.111 Type E- (gclug.) 5 1.699 1.100 0.497 0.253 0.135 0.128

Typc 0 (ATCC 33263) 0.5 0.093 Type F ( L n s h d ) 4 0.072

C. sporogabw (FDA-BIP) 0 0.081 c. p c f i g a u (PBP4) d 0.140 C. lcclni (ATCC 10779) d 0.083

C. novyi (ATCC 19402) d 0.101 C. butyricum (ATCC 8260) d 0.130

Canned Baby Clam spiked 5 0.849 0.562 0.536 0.161 0.120

Canned S.lmon spikd with 3 0.633 0.575 0.327 0.127

c.nned oystcr spiked with 3 0.683 0.520 0.259 0.073

Canned oyltsr spiked with 4 0.078

c.Msd CLm spiked with 4 0.093

Canned S.lmon rpiked 4 0.081

Canned CLm rpilred with 0 0.092

control' 1 0.085 2 0.074 3 0.081 4 0.073 5 0.116 6 0.105 7 0.053 8 0.050

WithtypeE-

W B

typeF

type Af

W Af

with type A'

C.sporogmrs

a Trypuukd culWfood prcpuat~on inoculated mth type E cdls. b Highest 10-fold dilution at which p i z i.p. injection (0.5 ml toxin sxtnct/monm) died c cOatrol1: Mab + conjug.ts+substnte. 2: Mab + toxinE + oooj. + rubs. 3: Mab + antirsrum + conj. +

subs. 4 Antismun + conj. + subs. 5: Mab + caokdmer th tb + amj. + subs. 6: Mab + TPGY broth + conj. + subs. 7: Conj. + subs. 8: Subs. done

dNotdstsrmioed. c Avenge of duplicate vdues loaded at 0.1 ml/well (a factor of 10 to MLD/d). Consolidated duplicate runs. f Types B aad F not detcmkd

conversion into toxoid for the immunization of BALB/c mice. The achievement in detecting type E toxin, at levels equivalent to 1-10 MLD/ml (5-50 pg/ml) in culture supernatants or in food extracts demonstrated a good relationship between

204 P.C.K. WONG

the biological activity of the toxins (MLD/ml) and the toxin-related-antigen con- tent of type E cultures. In this double-sandwich ELISA format, the Mab was used as the coating antibody to assure specific capture of the antigen; the rabbit anti- type E polyclonal antibodies as detecting antibodies which augmented the signal due to their abilities to combine with several epitopes on the same antigen molecule; the biotinylated labelled antibody as signal antibody and avidin-biotin complexes as amplifiers to immensely increase signal amplification. SuperBlock blocking buffer offered efficient blocking of residual sites on the microplate wells. Ultrafiltration using Filtron-SOK to desalt and clarify the analytes helped to reduce, if any, undesirable nonspecific background interference. However, in this pilot study, although much effort had been devoted to optimize the parameters used in this ELISA protocol, relatively few in-use test results were given. It is an- ticipated that more intensive tests may be underway to focus on testing, in replicates, varieties of natural or simulated swollen, leaker canned seafoods, smok- ed/cured fish products, fishes packed under modified atmospheres, and vacuum- packed cooked potatoes spiked with test botulinum/nonbotulinum clostridial organisms as well as other microorganisms indigenous to the products to demonstrate potential usefulness in this assay. Henceforth, more “real-life” samples can, therefore, be evaluated for sensitive, specific, and reliable results. Continued search for better plate blocking/storage reagents, means to increase sensitivity, and to speed up the ELISA procedure will also be pursued.

Strains of Clostridium butyricum have been reported to produce type E toxin causing infant botulism (Gimenez and Sugiyama 1988; Zhou er al. 1983). This interspecies toxin gene transfer was believed to be transduced by phage (Zhou et al. 1993). Whelan er al. (1992) sequenced the entire structural type E neurotoxin gene and revealed the genomic structures responsible for toxicity are well con- served among those of botulinum A, B, C, D, F, or tetanus toxins, while the antigenic determinants are not well conserved with highly divergent region cor- responds to the extreme COOH-terminus of each toxin. Conventional polyclonal antiserum tends to vary from lot to lot and contains a mixture of antibodies. Use of mouse or bioreactor for large scale Mab production makes it possible to pro- vide an infinite supply of test reagent with a predefined specificity. Finally, the 96-well microtiter plate format allows laboratory automation by using the ELISA work-station. Multiple samples for botulinum type E toxin detection can be analyzed in a single run with results obtainable in a much shorter time-frame, and in overall reduction of cost and labor.

ACKNOWLEDGMENTS

Thanks are due to Dr. Joseph L. Ferreira (FDA, Atlanta) and Don Smith (FDA, National Center for Toxicological Research) for providing the rabbit anti-type

E botulinum antiserum. Thanks are also due to Charles W. Noah (FDA, Dallas) for contributing the type F cultures, and Dr. J.L. Ferreira for confirming the type E Mabs’ specificity. Thanks are due to Emma Aranda, (FDA, Los Angles) for assistance in typing the manuscript, Dr. Debbie Johnson, Science Advisor, and Richard Ruby (FDA, Los Angeles) for reviewing a draft of this manuscript.

This research project was conducted under the Office of Regulatory Affairs, Division of Field Science Research Program, U.S. Food and Drug Administration.

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