Comp. Biochem. Physiol. Vol. 106B, No. 1, pp. 67-70, 1993 0305-0491/93 $6.00 + 0.00 Printed in Great Britain © 1993 Pergamon Press Ltd

MONOCLONAL ANTIBODIES NEUTRALIZING THE HAEMOLYTIC ACTIVITY OF BOX JELLYFISH (CHIRONEX

FLECKERI) TENTACLE EXTRACTS

S. P. COLLINS,* A. COMIS,t M. MARSHALL,* R. F. HARTWICK~ and M. E. H. HOWDEN*§ *School of Chemical and Biological Sciences, Deakin University, Geelong, VIC 3217, Australia (Fax 052 27-2001); tDeakin Research Ltd, University of Western Sydney, Richmond, NSW 2753, Australia; and :~Department of Marine Biology, James Cook University of Northern Queensland, Townsville, QLD 481 l,

Australia

(Received 26 January 1993; accepted 26 February 1993)

Abstract--1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri).

2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots.

3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under

any of our experimental conditions.

INTRODUCTION

The box jellyfish (Chironex fleckeri, Southcott) is a potentially lethal marine hazard of northern Australian waters which has been responsible for approximately 70 recorded fatalities (Sutherland, 1983). Extracts of the stinging tentacles of this cnidarian have been shown to possess distinct haemolytic and lethal activities with reported relative molecular masses of 70,000 and 150,000, respectively (Crone and Keen, 1969). These toxins have proved difficult to isolate because of their lability and their tendency to form aggregates (Endean, 1987; Comis et al., 1989). Olsen et al. (1984), Calton and Burnett (1986) and Naguib et al. (1988) have used immunoaffinity chromatography to isolate a number of toxic fractions from box jellyfish venom. Othman and Burnett (1990) have reviewed the techniques which have been found use- ful in attempting to purify box jellyfish venom toxins. The present study was undertaken to produce mono- clonal antibodies (MAbs) specific for the toxic com- ponents of box jellyfish tentacle extracts which would be useful in the analysis of these toxins. We report here the production of three MAbs which neutralize the haemolytic activity of box jellyfish tentacle ex- tracts.

§To whom correspondence should be addressed. Abbreviations--BCIP, 5-bromo-4-chloro-3-indoyl phos-

phate p-toluidine salt; ELISA, enzyme-linked immuno- sorbant assay; HAT, hypoxanthine, aminopterin, thymidine; MAb, monoelonal antibody; NBT, p-nitro- blue tetrazolium chloride; PEG, polyethylene glycol.

MATERIALS AND METHODS

Collection of jellyfish

Specimens of C. fleckeri were collected from the coastal waters of North East Queensland. The tenta- cles were frozen immediately and transported to the laboratory on dry ice (Comis et al., 1989).

Preparation of tentacle extract

Frozen tentacles were ground to a fine powder under liquid nitrogen in a mortar. The powder was allowed to thaw at room temperature and then centrifuged at 20,000g for 30 min at 4°C. The super- natant was passed through Sep-Pak C18 Cartridges (Waters Associates, Milford, MA) (Comis et aL, 1989). The filtrate was further passed through 0.8 #m cellu- lose acetate filters and 0.45/am nylon filters (Lida Manufacturing Corp., Bensenville, IL). This tentacle extract was stored as aliquots under liquid nitrogen.

Production of monoclonal antibodies

Mice were immunized with a 60% ammonium sulphate precipitate of tentacle extract. Eight-week- old female Balb/c mice were injected intraperitoneally (i.p.) with 50 #g of such precipitate in phosphate buffered saline (PBS) emulsified with Freund's com- plete adjuvant. The injection was repeated after 7 days and antibody titres examined by ELISA 7 days later. A final i.p. booster injection of 30/~g of the precipitate in PBS emulsified with Freund's incom- plete adjuvant was given 4 days prior to the pro- duction of hybridomas. Spleen cells from immunized mice were fused to NS/I myeloma cells at a ratio of 1:5 using a fusogen of 45% (w/v) PEG 4000 in distilled water containing 5% (v/v) dimethylsulphox- ide (Merck, Darmstadt, Germany) (De St Groth and

67

68 S . P . COLLINS et al,

Scheidegger, 1980). Hybridomas were selected by growth in HAT medium using murine peritoneal macrophages as feeder cells (Littlefield, 1964). Limit- ing dilution was used to clone hybridomas of interest two to three times (Goding, 1980). Hybridomas were cultured in Dulbecco's Modified Eagle's Medium supplemented with 1 mM glutamine, 50 I.U./ml peni- cillin, 50mg/ml streptomycin (Flow Laboratories, VIC, Melbourne, Australia) and 10% foetal calf serum (Commonwealth Serum Laboratories, VIC) in a humidified atmosphere of 95% air/5% C O 2 at 37°C. Ascites fluid was produced by i.p. injection of hybridomas into female Balb/c mice. Monoclonal antibodies were isotyped using a Mouse Typer tm Kit (Bio-Rad Laboratories, Melbourne, Australia).

Assay of neutralization of haemolytic activity

The assays of neutralization of the haemolytic activity of tentacle extracts were performed in the wells of 96-well microtitre plates. Equal volumes of mouse ascites fluid and a 1 : 100 dilution of tentacle extract in PBS were incubated for 30 min at room temperature. Fifty microlitres of this mixture was added to 200 #1 of washed mouse red blood cells. Haemolysis was recorded as the suspension in the wells became clear. Fifty microlitres of a 1:200 dilution of tentacle extract caused complete haemoly- sis in less than 60 secs.

Assay of neutralization of lethal activity

The lethal activity of tentacle extracts was assessed in adult Swiss mice (15-30g). Equal volumes of ascites fluid and a 1 : 100 dilution of tentacle extract in PBS were incubated for 30 min at room tempera- ture. One hundred microlitres of this mixture was injected into groups of three mice via a tail vein and death recorded within 5 min. One hundred microlitres of a 1 : 200 dilution of tentacle extract caused death in less than 20 sec.

Polyacrylamide gel electrophoresis and Western blotting

Polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970). Gels (15% acryl- amide) were run under three conditions: (1) reducing conditions, i.e. sodium dodecyl sulphate (SDS) gels with the sample prepared with SDS (2.5%), reducing agent (1% 2-mercaptoethanol) and heating (5min boiling water bath); (2) non-reducing conditions, i.e. SDS gels with the sample prepared with SDS but without reducing agent or heating; and (3) native conditions, i.e. gels and buffers without SDS and sample prepared without SDS, reducing agent or heating. Separated proteins of the tentacle extract were transferred to nitrocellulose membranes electro- phoretically (Beisiegel, 1986) and immunoprobed using the MAbs followed by anti-mouse Ig-alkaline phosphatase conjugate (Sigma Chemical Co., St Louis, MO). The blots were developed using

Table 1. Abilities of monoclonal antibodies to inhibit the lethal and haemolytic activity of box jellyfish tentacle

extracts

Anti- Monoclonal haemolytic Anti-lethal

antibody Isotype activity activity

4 M IgM Yes No 3 U IgM Yes No 7 A IgG2a Yes No

Mixtures of equal volumes of ascites fluid and a I:100 dilution of tentacle extract were incubated at room temperature for 30 min. They were then mixed with washed mouse red blood cells at room temperature to assess haemolytic activity or injected into adult mice to assess lethal activity.

BCIP/NBT substrate (Bio-Rad Laboratories, Aus- tralia).

RESULTS

Tentacle extracts produced by the method used in the present study have shown potent lethal and haemolytic activities (Comis et al., 1989). A 1:200 dilution of the extract caused complete haemolysis of mouse erythrocytes in vitro in less than 60 sec and death upon intravenous injection into adult mice in less than 20 sec. These extracts were used to coat the wells of microtitre plates in order to screen the hybridomas produced after immunization of mice with a 60% ammonium sulphate precipitate of tenta- cle extract. Those hybridomas displaying the strongest binding to extract were used to produce ascites fluid which was tested for anti-haemolytic and anti-lethal activity. Of those ascites tested, three were found to possess anti-haemolytic activity (Table 1). None of the MAbs tested displayed anti-lethal ac- tivity.

In order to establish to which component of the tentacle extract these MAbs were binding, the tenta- cle extract was subjected to reducing, non-reducing or native polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes and immunostained using the three MAbs. Table 2 shows that only two of the MAbs bound to a component of the blotted extract, and that this binding was only observed when the extract was electrophoresed under non-reducing conditions. Figure 1 shows that under non-reducing

Table 2. Abilities of monoclonal antibodies to bind to a component of Western blotted box jellyfish tentacle extract electrophoresed

under reducing, non-reducing and native conditions

Monoclonal Binding of monoclonal antibody to antibody Western blotted tentacle extract

Reducing Non-reducing Native conditions conditions conditions

4 M No Yes No 3 U No Yes No 7 A No No No

Approximately 100 ,u g of tentacle extract was electrophoresed under reducing, non-reducing and native conditions and transferred to nitrocellulose membranes. The membranes were then exposed to monoclonal antibodies followed by anti-mouse Ig-alkaline phosphatase conjugate and developed.

Box jellyfish haemolytic toxin 69

I 2 3 4

9 7 . 4 k

42 .7k

2 I.Sk ,.=*'

Fig. 1. Binding ofmonoclonal antibodies to a component of Western blotted box jellyfish tentacle extract elec- trophoresed under non-reducing conditions. Approximately 100 #g of tentacle extract was electrophoresed under non-re- ducing conditions (SDS-polyacrylamide gels with a sample prepared with SDS but without reducing agent or heating) and transferred onto nitrocellulose membranes. The mem- branes were then exposed to monoclonal antibody followed by anti-mouse Ig-alkaline phosphatase conjugate. Lane 1: Coomassie Blue-stained polyacrylamide gel of tentacle ex- tract. Lane 2: Western blot of lane I probed with MAb 4 M. Lane 3: Western blot of lane 1 probed with MAb 3 U. Lane

4: Western blot of lane I probed with MAb 7 A.

conditions these two MAbs bound to the same tentacle extract component (mol. wt approx. 50,000). When the tentacle extract was subjected to electro- phoresis under reducing conditions before blotting, none of the MAbs displayed binding. Similarly, when the extracts were electrophoresed under native con- ditions, no binding by MAbs could be detected.

DISCUSSION

Extracts of the tentacles of the box jellyfish have been reported to possess lethal, haemolytic, myotoxic and dermatonecrotic activities (Baxter and Marr, 1969; Endean, 1987). The haemolysin in these ex- tracts was reported to have a relative molecular mass of approximately 70,000 (Crone and Keen, 1969) and of being proteinaceous in nature (Marr and Baxter, 1971). It has also been reported as requiring an intact disulphide bond and the presence of a divalent cation for maximal activity (Crone, 1976). The haemolysin has also been shown to interact with lipid monolayers and to be inactivated by gangliosides (Keen, 1973; Crone, 1976). These observations are consistent with

the box jellyfish haemolysin being a relatively large protein with some hydrophobic properties and a tertiary structure stabilized by at least one disulphide bond.

In the present study we have described three MAbs which inhibit the haemolytic activity of box jellyfish tentacle extracts. Two of these MAbs bind to an extract component of approximately 50,000 relative molecular mass. If we assume that this component is the haemolytic toxin, then this electrophoretic esti- mation of relative molecular mass is lower than the reported chromatographic estimation of 70,000 (Crone and Keen, 1969). This difference may be due to the different natures of these estimation techniques and the effects of the charge and shape of the molecule under chromatographic conditions.

Table 2 shows that the binding of the MAbs to a Western blotted tentacle extract component varied with the clectrophoretic conditions used. Under re- ducing conditions no binding could be demonstrated. This observation is consistent with the epitopes re- quired for binding being destroyed by reduction and/or heating. Under non-reducing conditions, two of the three MAbs (4 M and 3 U) which inhibited haemolytic activity (Table l) bound to a venom extract component of 50,000 relative molecular mass. The third monoclonal antibody (7 A) did not bind when the extract was electrophoresed under non-re- ducing conditions. If we assume that 7 A is specific for the same component, as are 4 M and 3 U, this suggests that the specific epitope for 7 A is destroyed in the presence of SDS detergent. Alternatively, 7 A may be specific for a different component of tentacle extract which is required for haemolytic activity and whose specific epitope is similarly destroyed by the presence of detergent.

None of the three anti-haemolytic MAbs bound to Western blotted tentacle extract components when the extract was electrophoresed under native con- ditions. This result was suprising since, under these conditions, the tentacle extract components should be in their native state and better able to bind the antibodies. It may be that in the absence of detergent the component of 50,000 relative molecular mass forms aggregates and fails to enter the gel. The propensity of box jellyfish toxins to aggregate has been reported previously (Olsen et al., 1984). The inability of the MAbs tested to neutralize the lethal activity of whole tentacle extract may be due to the presence of lethal toxin(s) in the tentacles which are not haemolytic, and which are not therefore neutral- ized by such MAbs. Crone and Keen (1969) and Comis et al. (1989) detected such non-haemolytic toxins in tentacle extract.

In conclusion, we have produced three MAbs which inhibit the haemolytic activity of box jellyfish tentacle extracts in vitro. Two of these MAbs display specific binding to a component of 50,000 relative molecular mass in these extracts on Western blots. We hope that one or more of the MAbs will prove

70 S.P. COLLINS et al.

useful for the immunoaffinity purification and characterization of the haemolytic toxin.

Acknowledgements--This work was supported by the National Health and Medical Research Council of Australia and Deakin University. We thank Dr M. Brandon and Deakin Research Ltd for generous assistance with mono- clonal antibody techniques.

REFERENCES

Baxter E. H. and Marr A. G. M. (1969) Sea wasp (Chironex fleckeri) venom: lethal, haemolytic and dermatonecrotic properties. Toxicon 7, 195-210.

Beisiegel U. (1986) Protein blotting. Electrophoresis 7, 1-18. C_alton G. J. and Burner J. W. (1986) Partial purification

of Chironex fleckeri (Sea wasp) venom by immunochro- matography with antivenom. Toxicon 24, 416-420.

Corals A., Hartwick R. F. and Howden M. E. H. (1989) Stabilization of lethal and hemolytic activities of box jellyfish (Chironexfleckeri) venom. Toxicon 27, 439--447.

Crone H. D. (1976) Chemical modification of the haemolytic activity of extracts from the box jellyfish Chironex fleckeri (Cnidaria). Toxicon 14, 97-107.

Crone H. D. and Keen T. E. B. (1969) Chromatographic properties of the hemolysin from the cnidarian Chironex fleckeri. Toxicon 7, 7947.

De St Groth S. F. and Scheidegger D. (1980) Production of monoclonal antibodies: strategies and tactics. J. Immunol. Meth. 35, 1-21.

Endean R. (1987) Separation of two myotoxins from nema- tocysts of the box jellyfish (Chironexfleckeri). Toxicon 25, 483-492.

Goding J. W. (1980) Antibody production by hybridomas. J. Immunol. Meth. 39, 285-308.

Keen T. E. B. (1973) Interaction of the hemolysin of Chironex fleckeri tentacle extracts with lipid monolayers. Toxicon 11, 293-299.

Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680--685.

Littlefield J. M. (1964) Selection of hybrids from mating fibroblasts in vitro and their presumed recombinants. Science 145, 709-710.

Marr A. G. M. and Baxter E. H. (1971) Effect of proteolytic enzymes on the venom of the Sea wasp Chironexfleckeri. Toxicon 9, 431-433.

Naguib A. M. F., Bansal J., Calton G. J. and Burnett J. W. (1988) Purification of Chironex fleckeri venom com- ponents using Chironex immunoaffinity chromatography. Toxicon 26, 387-394.

Olsen C. E., Pockl E. E., Calton G. J. and Burnett J. W. (1984) Immunochromatographic purification of a nematocyst toxin from the cnidarian Chironex fleckeri (Sea wasp). Toxicon 22, 733-742.

Othman I. and Burner J. W. (1990) Techniques applicable for purifying Chironex fleckeri (box jellyfish) venom. Toxicon 28, 821-835.

Sutherland S. K. (1983) Australian Animal Toxins, pp. 359-373. Oxford University Press, Melbourne.