Bmlogzcals ( 1991) 19, 293-298

Development of an Enzyme Immunoassay for the Detection of Clostridium novyi Type B Alpha Toxin E. Pietrzykowski, J. Cox, M. Zachariou and A. MacGregor R & D Diwslon, Commonwealth Serum Laboratones Limited, Parkvflle, Victona 3052, Austraha

Abstract. Clostndium novyitype B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoJded and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can ehmmate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological actwlty are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoidmg and vaccine formulation.

Introduction

Materials and methods

Clostridium novyt type B produces several exogenous proteins including an alpha toxin which causes a fatal condition m sheep and cattle known as Black disease. The disease can be controlled effectively by vaccination of animals with alpha toxoid. 1 The alpha toxin from C. novyi type B has been partially purified and reported to be a protein with a molecular weight of 132 kD. 2 Alternatively, a molecular weight of 260 to 280 kD for the purportedly identical toxin from C. novy~ type A has been reported. 3,4 Most recently, type A alpha-toxin has been reported to have a molecular weight of 200 kD. 5 The procedure specffied by the British Pharmacopoeia for the quantitative estimation of C. novyt alpha toxin and toxoid is a biological assay. 6 This test is labour-intensive, takes 4 days and requires large numbers of mice. For these reasons, an in vitro test would be preferred. Here we describe the purification and characterization ofC. novyz alpha toxin and the development of an enzyme immunoassay (EL&), based on two noncompeting monoclonal antibodies (MAbs), suitable both as an in-process test for monitoring toxin and toxoid levels and recoveries and as a subsequent quality control identity test. The assay is specific and, even in a 5 min format, more sensitive than the mouse lethal challenge assay.

Punhcahon of alpha toxin The toxin was purified by HPLC from the supern a t a n t fluid of a fermenter grown C. novy~ broth culture. Cells and debris were removed by centrifugation at 5000 g for 1 h and the s u p e r n a t a n t concentrated two-fold on a YM 100 (Amicon Corporation, Massachusetts, U.S.A.) membrane, and washed against 100 mM sodium phosphate buffer pH 7-0. Solid NaC1 was added to the concentrate to a final concentration of 1 M, and the sample was then loaded onto a Phenyl-Sepharose column (low substitution Fast-Flow, Pharmacia, Uppsala, Sweden) pre-equilibrated with 100 mM sodmm phosphate buffer pH 7-0 containing 1 M NaC]. The column size was 140 x 30 mm and flow rate was 3 ml/min for loading and elution. The eluate containing the toxin was concentrated 200-fold on a YM 100 membrane, dialysed against 20 mM Tris/HC1 buffer pH 7.0 and loaded onto a Mono Q anion exchange column (HR 5/5 Pharmacia, 5 × 50 mm) which had been pre-equilibrated with Tris/HCl. Elution was carried out on a Waters HPLC system using a gradient generated over 51 min starting with Tris/HC1 and finishing with Tris/HCl containing 0.5 M NaCl. The Mono Q fractions containing the toxin were concentrated 200-fold on a YM 100 membrane and loaded on to a Superose 12 column (S-12 HR Pharmacia, 300 x 10 mm) pre-eqmlibrated with

1045-1056/91/040293+06 $03 00/0

© 1991 The International Assomatlon of BmloglcalStandard~zatmn

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phosphate buffered saline (PBS) pH 7.1 which was also used for elution. The bioassay was used to detect alpha toxin. Purified toxin (1.5 mg/ml) was toxoided with formaldehyde at a final concentration of 0.15% w/w in the presence of 0.05 M lysine HC1. After 4 weeks at 4°C, the resultant toxoid was used both as antigen for immunization of mice and as capture antigen in an EIA for the identification of monoclonal antibodies.

Product/on of hybndomas Hybridomas secreting antibody to C. novy~ alpha toxin were produced by fusing either lymph nodes (pophteal and inguinal) or splenocytes with SP2/0Agl4 cells. Female Balb/c mice, (6-8 weeks old) which were used for splenic fusion were injected (i.p) with 0.5 ml toxoid (10 pg/mouse) emulsified in complete Freund's adjuvant. F u r t h e r inoculations (i.p) of toxoid in Freund's incomplete adjuvant were given on days 21 and 35. Retro-orbital bleeds were obtained from anaesthetized mine for determination of antibody levels. The development of specific anti-toxin antibodms in mice was monitored by an EIA as described below. When required for fusion, the animal with the highest serum antibody level was boosted (iv) with 5 pg oftoxoid 5 days prior to the fusion. For the lymph node fusion, 5 Balb/c mice were inoculated in one rear footpad with 50 pl containing 5 pg toxoid emulsified in complete Freund's adjuvant. 7 All other procedures were as described previously s Cells from wells with specific antibody were cloned twice by limiting dilution on 3T3 Balb/c feeder layers in 96-well plates. Initial selectton by EIA of clones recognizing C novyt Polystyrene plates (Maxisorb/NUNC, Denmark) were coated overnight at 4°C with toxin or toxoid (2 pg/ml) in 0.05 M bicarbonate buffer pH 9.6 then washed five times in PBS containing 0.05% Tween 20 (PBSTween). Unreacted binding sites were blocked by incubation for 1 h with 1 mg/ml casein at room temperature. The diluent used in all assays was Blue Diluent (CSL, Australia), a PBS Tween diluent containing casein. Test samples were incubated for 30 min at 37°C (0-1 ml/well), washed as before with PBS Tween then similarly incubated with horse radish peroxldase (HRP)---conjugated goat anti-mouse IgG-gamma chain specific (KPL, Maryland, U.S.A.). Peroxidase activity was measured by addition of 0.1 ml/well of a substrate solution containing H202 and tetram-

ethylbenzidine. 9 After 5 min at room temperature, the reaction was stopped by addition of 0.05 ml/well 0.5 M H2804. Absorbance readings were made at 450 nm on an automated EIA plate reader.

Immunoblott/ng The purified toxin was subjected to SDS polycrylamide gel electrophoresis (Pharmacia Phast Gel System, gel gradient 4-15%). Western blots were prepared by direct transfer to nitrocellulose film using the same system. Molecular weight markers were included m each separation. After blocking of unreacted binding sites with a 5% (w/v) solution of casein, the nitrocellulose was cut into strips and test samples containing monoclonal antibodies were allowed to react with individual strips for 1 h at 37°C then unreacted antibodies were removed by washing and HRP-labelled goat anti-mouse IgG (KPL) was added for 1 h at 37°C. The antigen-antibody bands were located with 4-choro-l-naphthol reagent (KPL). Mouse lethality test Hybridoma lines producing monoclonal antibodies against toxin (as detected by EIA) were tested by both a routine C. novyz L+/4 antitoxin assay to detect neutralization and by a precipitation test to determine whether monoclonal antibodies were able to recognize and bind toxin in solution. The routine L+/4 antitoxin assay s was used to determine if monoclonal antibodies possessed toxin-neutralizing activity. Briefly, doubling dilutions of ascites from 64 to 1 pg/ml were incubated with one L+/4 dose of alpha toxin for 60 min at 37°C then 0.5 ml injected intravenously into mice. Mine were observed for 72 h and deaths recorded. The alpha toxin used in this assay was standardized against the International Standard for Gas Gangrene Antitoxin (Clostridium novyi). For the precipitation test, all incubations were performed in 0.5% w/v peptone saline adjusted to pH 8-0 to maximize binding of immunoglobulins by protern A. Dilutions of the crude C. novy~ toxin containing 1, 2, 4, 8, 16 and 32 LDs0 were incubated with monoclonal antibodies from culture supernatants {concentration of antibodies was adjusted to 5 pg/ml) for 30 mm at 37°C. Rabbit anti-mouse lmmunoglobuhn (Dako, Denmark) was added to a final concentration of 5 pg/ml and incubation continued for a further 30 m m at 37°C. Protein A sepharose CL-4B was then added and after incubation for 10 min, mixtures were centrifuged (1000 g, 2 min) to pellet the antigen-antibody complex. Samples of s u p e r n a t a n t

Detection of C. novyhtype B alpha toxin

were injected i.v. into mice (0-5 ml dose). Death or survival of mice over a 72 h period was used as the indicator of the ability of the monoclonal antibody to recognize toxin in solution.

Protein A punhcat~on of monoclonal ant~bodles Ascitic fluid was produced by injecting hybridomas (5 x 105 cells/mouse) i.p. into pristane-primed mice. Ascites pools were clarified by centrifugation at 1500 g for 10 min, filtered through a 0.2 pm membrane and purified by Protein A affimty chromatography, z° The filtrate was adjusted to pH 8.0 with 3 M Tris buffer and passed through a Protein A Sepharose CL-4B column (Pharmacia, Uppsala, Sweden). The column was then washed with 0-1 M phosphate buffer pH 8.0 until the optical density at 280 nm of the eluate was less t h a n 0.05. The mouse IgG1, lgG2a and IgG2b were eluted with 0.1 M citrate buffer pH 5-5, 4.5 and 3.5 respectively.

Preparation of F(ab l)2 fragments from IgG 1 Protein A purified IgG1 (prepared as described above) from hybridoma line CN9.7B9 was dialysed against 0.1 M sodium citrate buffer pH 3.5 overnight at 4°C. Pepsin agarose (Sigma) was added to the IgG solution (112.5 U pepsin/ml IgG) and mixed on a rotary mixer for 5 h at 37°C. At the end of the incubation the pH was adjusted to 7.5 by addition of 3 M Tris buffer pH 8 2. The pepsin agarose was removed by centrifugation (2000 g for 2 min) and the final preparation was dialysed against PBS overnight. The purified mouse IgG and pepsin-cleaved fragments were analysed by SDSPAGE under non-reducing conditions (PhastSystem, Pharmacia).

Screening of hybrldomas by EIA F(ab I)2 fragments coated to solid phase To search for a second monoclonal antibody which could recognize a d~fferent site on the toxin, the EIA was modified as follows: purified F(abl)2 fragment (derived from hybridoma line CN9.7B9) was coated to the plate at 10 pg/ml as a capture antibody. Wells were blocked with casein as described previously then the optimal dilution of crude toxin (150 ng/ml in Blue Diluent) was added, incubated for 30 min at 37°C and the plates washed thoroughly with PBS/Tween. The standard EIA procedure as described previously was followed except that the conjugate used was an HRP labelled anti mouse IgG (7 chain-specific) (Sigma Missouri, U.S.A.). Serum samples taken at time of fusion and cell culture

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medium were used as positive and negative controls respectively.

Preparation of HRP conjugates Antibodies were conjugated to HRP according to published procedures. 11

EIA assays for the detection of the toxln/toxold For detection of toxin/toxoid the EIA was modified as follows. The purified IgG1 from CN9.7B9 hybridoma hne was coated to the plates at 2 pg/ml. The plates were blocked, stabilized, dried, sealed and stored at 4°C. Samples and controls were diluted as appropriate in Blue Diluent. In the rapid simultaneous EIA, samples were added to the wells coated with capture antibody (CN9.7B9) at the same time as the HRP-conjugated second antibody. Following a 5 min reaction at room temperature, wells were washed and substrate added. Blue colour development could be read visually after several minutes or alternatively on a plate reader after addition of H2SO 4. In the sequential EIA system, the samples were added to the plates coated with capture antibody and incubated for 30 min at 37°C. After washing, HRP-conjugated second antibody was added and incubated for a further 30 min at 37°C. Substrate was added and the result read on a EIA plate reader after addition of H2SO 4. Results

Purlhcatlon of alpha toxin Figure 1 shows a typical purification of alpha toxin. The crude alpha toxin was partially purified by hydrophobic chromatography on a column of Phenyl-Sepharose. The toxin was found in a peak eluted with 0-6 M NaC1 in sodium phosphate buffer pH 7-0. Fractions identified as containing toxin (indicated by the arrow) were combined, concentrated 200-fold and applied to a Mono Q column. The toxin from this column was eluted at 33 min with a NaC1 gradient from 0 to 0.5 Min Tns-HC1 buffer. The final purification was performed on a Superose 12 column. The toxin was eluted after 28.6 min at a flow rate of 0.4 ml/min. When analysed by sliver-stained PAGE, the purffied material showed a single band of molecular weight around 200 kD. The minimum mouse lethal dose of this purified alpha toxin was 4 ng.

Hybndoma selection Spleens from three mice (CN8, CN9 and CN10) and lymph nodes from 10 mice (CN13 and CN18)

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Figure 1. Purificatmn of C. novyt alpha-toxin. Arrows (e-~) indicate fractions with toxin actwlty (a) Elutmn profile on Phenyl Sepharose (b) Elutmn profile of toxin peak from (a) on Mono-Q (c) Elution profile of toxin peak from (b) on Superose 12. were used m this study (Table 1). Considering the four fusions (CN8, CN9, CN10 and CN13), where selection was based on recognition of toxin or toxoid on an EIA plate, from the total of 1920 wells seeded, 1353 (70%) yielded hybridomas. From these, 12 stable hybridoma hnes (0.9%) were identified and isolated. Eleven of these 12 hybridoma lines were further characterized to determine their usefulness for the estabhshment of an EIA specific to novyi toxin and/or toxoid The twelfth line, because it was of IgM lsotype, was not characterized further. It can be seen that three of these 11 recognized toxin by Western blot but only one showed strong binding to toxin in solution (at least eight-fold reduction in lethality following immuno-precipitation). Additionally, a further four monoclonal antibodies

showed weak binding to novyi toxin in solution (a two-fold reduction m lethality). It is interesting to note t h a t the strongly binding antibody and two of the four weakly binding antibodies recogmzed toxin by Western blot. None of these antibodms was able to neutralize the toxicity of novyi toxin when tested in the routine L + /4 toxin neutralization assay. Fusion CN18 was undertaken to find a non-competing partner for the monoclonal antibody identified above whmh strongly bound toxin in solution. To achieve this the F(ab'),, portion of the antibody was bound to an EIA plate, reacted with toxin then this bound toxin used as the capture phase for screening the new family of hybridomas. Three hybridoma hnes were identified in this way. Significantly, all three recognized novyi toxin by Western blot and

T a b l e 1. Identification and characterization of monoclonal antibodies raised against novyi toxoid. Number recognizing toxin Number of wells Fusion

Route of immumzation

Seeded

CN 8 CN 9 CN 10 CN 13 Total CN 18

i p. i.p. i.p. Footpad -Footpad

480 480 480 480 1920 600

With hybridomas 480* (100%) 341 " (71%) 375":: (78%) 157" (33%) 1353 (70%) 598t (100%)

Number of stable lines/isotype

In solution By Western blot

1 IgG1 5 IgG1 1 IgG1 5 4 × IgGl$ -3 1 × IgG2a 2 × IgG2b

1 2 --3 3

Strong

Weak

1 --1 3

1 2 -1 4 --

'~Hybndoma supernatants screened against toxin or toxoid coated to an EIA plate. t Hybridoma supernatants screened against toxin captured by the Fab 2 fragment derived from CN 9-7B9. $ A fifth hybndoma, of IgM isotope, was not further characterized.

Detection of Conovyi: type B alpha toxin

strongly bound the toxin in solution. However, none was able to neutralize toxin activity in w v o .

Detection of novyt toxin by EIA Each of the monoclonal antibodies identified in the final fusion (CN18), when labelled with HRP and used in the rapid assay format for novyi toxin, resulted in an assay with a sensitiwty of at least 2 ng toxin/ml. Extension to a 1 h sequential assay increased assay sensitivity four-fold. Thus, even m the less sensitive format, the assay could detect less than one MLD of novyi toxin. Discussion

The EIA developed here is a highly sensitive and rapid assay for quantitation of novyi toxin and toxoid. In addition, being based on monoclonal antibodies, it is of high specificity. The assay therefore offers an alternative to the mouse bioassay for the estimation of antigen content during fermentatmn, toxoiding and vaccine formulation. Because of its speed and sensitiwty it can also be used to monitor toxin production during fermentation so t h a t fermentation can be terminated at the optimal time. An initial concern in the development of this assay was to be certain t h a t the purified toxin was indeed alpha toxin rather than some other protein with a low level of alpha toxin contamination. This concern was increased by the difference in the molecular weight observed by us and t h a t reported to t h a t time by other workers. Initial confirmation was obtained by determination of the mouse minimum lethal dose (MLD) for our purified toxin. The figure of 4 ng was less than previously described. 4 Subsequent confirmation was achieved when several of the monoclonal antibodms were shown by the precipitation test to remove the toxic activity from a solution containing novyi toxin. Two procedures were used here to select useful hybridomas. In the first procedure, toxin and toxoid coated to EIA plates were used as the capture phase for selection of monoclonal. As a result, although an acceptable percentage of stable hybridoma hnes was isolated (12 from 1353 or 0.9%), only one line (CN9 7B9) was able to recognize the toxin molecule in solution. We therefore assume t h a t most epitopes presented by novyi toxin bound by hydrophoblc forces to a polystyrene support are non-native epitopes. In the final fusion, hybridomas were selected using as the capture phase novyi toxin bound to mouse F(ab')., rather than directly to polystyrene. The intention was to select a monoclonal antibody suit-

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able as a partner to CN9 7B9 in a sandwich EIA for toxin. Three stable lines resulted (0.5%), all of which recognized a native epitope on the toxin molecule. The selection procedure therefore greatly increased the probability of obtaining a monoclonal antibody of the required specificity even though the percentage of stable hybridomas isolated was, m fact, less. Similar results have been observed by Macfarlan (pers comm) when selecting monoclonal antibodies to h u m a n interferon y. The molecular weight of Clostr~d~um n o v y i type B alpha toxin reported here varies significantly from t h a t reported by earlier workers for alpha toxin from type A 2 and type B 3,4 C. n o v y t but is in agreement with the recent work of Mauler et al. 5 based on slabgel SDS-PAGE analysis. Izuml et al. 4 estimated the molecular weight from both SDS-PAGE and Sephadedx G-200 gel filtration measurements. For their SDS-PAGE measurements, inaccuracies may have been introduced both by their use of tube gels which would introduce a sample to sample variation and by the fact t h a t their highest molecular weight marker was y-globulin (molecular weight 150 000), thus necessitating a considerable extrapolation. Similarly, a careful examination of their gel filtration data shows that the alpha toxin eluted very close to catalase (molecular weight 230 000). In our studies, a slab gel was used and the alpha toxin was found to migrate only marginally slower than rabbit muscle myosin (molecular weight 200 kD) thus supporting our estimate of 200 kD. The mouse minimum lethal dose of alpha toxin purified by us was 4 ng. This is considerably less t h a n the 17 ng reported by earlier workers 4 but in close agreement with the figure of 6 ng reported by Mauler et al. 5 It would seem reasonable to suggest the toxin reported in this earlier work was either less pure or had suffered considerable inactivation during or subsequent to purification.

Acknowledgements We t h a n k Chris Kapouleas Karampetsos for valued assistance.

and

Peter

References

1. Blood DC, Radostlts OM. Veterinary Medicine, 7th edn London: Bailliere Tmdall, 1989 2 Phllhps AW, Batty I, Wood RD A partial characterlsation of the a toxin of Clostrtdtum oedematiens type B. Eur J Blochem 1970, 14: 367-371. 3 Izuml N, Nnro M. Kondo H Clostr, dlum oedemattens type A toxin. The correlation between the lethal and edematmmg actwities. Jap J Med Sc~ Blol 1983; 36. 67-744

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4. Izumi N, Kondo H, Ohishl I, Sakaguchi G. Purification and charactensation of a toxin of Clostndtum oedemattens type A. J a p J Med Sci Biol 1983, 36:135-145 5 Mauler F, Bette P, Habermann E. Alpha-toxin of Clostrtdlum novyl type A: Purification, iodination and binding properties. Zbl Bakt 1990; Suppl 19: 185-186. 6. Brutish Pharmacopoeia (Veterinary). London: Her Majesty's Stationery Office, 1985; AppendLx XIVBAl13. 7. Mirza IH, Wilkm TJ, Cantarini M, Moore K. A comparison of spleen and lymph node cells as fusion partners for the raising of monoclonal antibodms after different routes of lmmumsation. J Immunol Meth 1987; 105:235-243 8. MacGregor A, Kormtschuk M, Hurrell JGR, Lehmann NI, Coulepis AG, Locarnini SA, Gust ID. Monoclonal antibodies against Hepatltm A virus. J Clin Micro 1983; 18. 1237-1234.

9. Bos ES, van der Doelen AA, van Rooy N, Schuurs AHWM 3, 3'5, 5'-tetramethylbenzidme as an Ames test negative chromogen for horse-radish peroxidase in enzyme immunoassay. J Immunoassay 1981; 2: 187-204. 10. Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A Sepharose. Immunochem 1978; 15' 429-436. 11 Wilson MB, Nakane PK. Recent developments in the periodate method of conjugating horseradish peroradase (HRPO) to antibodies. In Knapp W, Holubar K, Wick G, eds Immunofluorescence and Related Staining Techniques. Amsterdam: North-Holland Biomedical Press, 1978: 215-224.

Recewed for pubhcation 18 March 1991; accepted 24 May 1991.

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin.

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophore...
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