Production and partial characterization of monoclonal antibodies to Bothrops asper (terciopelo) myotoxin

Tanlc»~, Vol. Z6, No. 7, pp. 673-689. 1988 . Prinoed in Q~at arlddn .

0041-0101/88 53.00+ .00 ® 1988 Perpmon Proe pk

PRODUCTION AND PARTIAL CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO BOTHROPS ASPER (TERCIOPELO) MYOTOXIN BRUNO LOMONTE I and LAWRENCE KAHAN Z 'Instituto Clodomiro Picado, Facultad de Microbiologia, Universidad de Costa Rica, San José, Costa Rica, and =Department of Physiological Chemistry, University of Wisconsin-Madison, Wisconsin 53706, U .S .A .

(Amepted for publication 28 January 1988) B . LoMOxrE and L . Kwtt~lv. Production and partial characterization of monoclonal antibodies to Bothrops riper (terciopelo) myotoxin . ToxJcon 26, 675 - 689, 1988 . - Seven marine monoclonal antibodies against Bothrops aspen myotoxin were produced and partially characterized. They revealed the presence of at least four cross-reacting basic components in crude venom, with a common subunit mol . wt of 16,000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis, but alight differences in charge. These myotoxin-related components might be isoforms of the toxin . By Western blotting and enzymo-immunoassay binding techniques, differences in the reactivities with basic venom fractions were observed among monoclonal antibodies, suggesting differences in epitope specifidties . Three antibodies aces-reacted with B. nummUa crude venom . Two monoclonal antibodies were utilized to develop a two-site enzymo-immunoassay for myotoxin detection at the nanogram level . Three of the antibodies (one IgM and two IgG,) showed ability to neutralize myotoxiclty of purified myotoxin in preincubation type experiments.

INTRODUCTION

Of (oral myonecrosis in the development of sequelae following snakebite envenomation has led to an interest in the study of myotoxins (OWNBY et al., 1976 ; CHINZEI, 1979; FABIANO and Tu, 1981 ; HARRIS and MACDONELL, 1981 ; MEBS et al., 1983; GUTi13RREZ et al., 1984, 1986a; MEBS and $AMEJIMA, 1986), and their neutralization by antibodies (OWNBY et al., 1983, 1986; MEBS, 1986; BOBER et al., 1987 ; LOMONTE et al., 1985; 1987a) . The for-de-lance (Bothrops aspor), commonly known in Costa Rica as `terciopelo' is the most important snake species in Central America from the medical point of view (BOLAI~os, 1982) . A myotoxic phospholipase A has been isolated from its venom (GUTI>3RREZ et al., 1984 ; MEBS and SAMEIIMA, 1986) and shown to play an important role in the myotoxic activity of crude venom (LOMONTE et al., 1985, 1987a) . In the present work, the production and some characteristics of a set of monoclonal antibodies (MAbs) to B. aspen myotoxin, are described. In addition, some applications of these antibodies are explored, such as the development of a sensitive enzyme-immunoassay (EIA) for myotoxin detection, and the immunoaffinity-purification of myotoxin . THE IMPORTANCE

MATERIALS AND METHODS Venom jractionatton and isolation of myotoxin

B. aspor venom (Instituto Clodomiro Picado, San José, Costa Rica) was fractionated on CM~ephadex C-25 (Pharmacia Fine Chemicals, Uppsala, Sweden) as described by GtrrlBesez et al . (19866) . The peak 675

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B . LOMONTE and L. KAHAN

corresponding to myotoxin (peak S) was recycled under identical conditions, and its purity was evaluated by 12ßb polyacrylamide gel electrophoresis in the presence of urea (urea-PAGE) using the cathodic system of TtenuH et al . (1971), and by sodium dodaylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the procedure of Lw$huaa (1970) . Immunization Four BALB/c mice (virus free ; Sprague-Dawley, Indianapolis, IN, U.S .A .) of 8 weeks of age were immunized by i.p . injection of SO Ng of native myotoxin emulsified in 300 pl Freund's complete adjuvant (Calbiochem, Saa Diego, CA, U.S .A .) . Booster i.p . igjations of 20 Ng of myotoxin in saline were given 15, 30 and 45 days after priming. Seven days later, individual serum antibody responses were evaluated by EIA, and the best responding animal was related as donor of spleen cells. Four days before fusion, the mouse received 20 Ng of myotoxin in 1S0 pl of 0.15 M NaCI by i.v . route, and 3 and 2 days before, 20 Ng in 100 NI of saline by i.p . route. Production of anti-myotoxin hybridomas Spleen cells were fused with 1 x 10' P3/NSI/1-Ag4-1 myeloma cells (NS-1 ; ATCC, Rockville, MD, U.S .A .; Köxt.ett and M~LSrEnv, 1976), usingpolyethylene glycol 1500 (Boehringer-Mannheim, Indianapolis, IN, U .S .A .) according to the manufacturer's instructions . Cells were distributed in 30 96-well plates (Corning, New York, NY, U.S.A .) in HAT medium (Dulbaco's Modified Eagle medium with 6 x 10 -' M hypoxanthine, 2 x 10 -' M thymidine, and S x 10 -' M amethopterin) with 20°ío fetal bovine serum (Gibco, New York, NY, U.S .A .), and cultured at 37°C with 7fo CO=. Feeder cells consisted of either O.S44 (v/v) mouse blood cells (20 plates) or sheep blood cells (10 plates). Supernatants were screened for anti-myotoxin antibody by EIA, and positive colonies were cloned by limiting dilution in 96-well plates at 0.3 and 0.1 cells/well . Final clones were considered statistically valid if less than 9 wells/plate had colonies (Poisson probability of single colony > 9S%i ; DE Bras et al., 1983) and if a single colony was observed microscopically. Enyyme-immunoassay Flat bottom polystyrene plates (Linbro-~tertek EIA plates, Flow Laboratories, McLean, VA, U.S .A .) were coated with 0.4 pg/well of myotoxin in 0.1 M Tris, 0.15 M NaCI, pH 9.0 buffer . This toxin concentration was selected from tests ova the range of 0.05-8.0 Ng/well, using a rabbit anti-myotoxin serum (LOMONTE et al., 1987a). All samples (100 ~1/well) were incubated overnight at 4°C. When naessary, dilutions were prepared in culture medium . After five washes with buffer A (O.OS M Tris, 0.15 M NaCI, 1 .0 mM MgCI=, 20 NM ZnCI,, S mM NaN pH 7.4) plates were blocked with SO% bovine serum in buffer B (0 .2Sßh gelatin in buffer A) for 10 min. The serum was flicked out and 100 pl/well of conjugate was added for 2 hr at room temperature . This consisted of 2S ng/ml affinity-purified goat anti-mouse IgG (H+L) conjugated to alkaline phosphatase (Kierkegaard 8c Perry, Gaitherrburg, MD, U.S .A .) in 10ß'o serum in buffer B. After washing, 200 pl/well of 660 ~M 4-methyl-umbelliferyl phosphate (sodium salt ; Research Organics, Cleveland, OH, U.S .A .) in 1 M 2amino-2-methyl-l-propanol, 25 ~M ZnCI=, 1.0 mM MgCI pH 10.3 was added, and fluorescence was measured at 0, 15, 30, 45 and 60 min in a Dynatah Microfluor Reader (Alexandria, VA, U.S .A .) or a Titertek Fluoroskan (Flow Laboratories). Readings at time 0 were subtracted from the final readings (usually 60 min) in order to eliminate the background signal originating from 4-mtthylumbelliferone initially present in the substrate. As positive controls, either mouse or rabbit anti-myotoxin sera were utilized . Culture medium, normal rabbit or mouse sera, or a commercial marine ascitic fluid (P3x 63 BALB/c myeloma, > 10 mg/ml IgG of unknown spaificity ; BRL, Gaithtrrburg, MD, U.S .A .) were utilized as negative controls . In all experiments, a sample was considered positive if readings (as arbitrary fluorescence units) were at least three times higher than controls . Production of MAbs Hybridoma ce)IS (2 x 10~ were injected i.p . into BALB/c mice, primed 12 days earlier with O.S ml of 2,6,10,14-tetramethtylpentadaane . MAbs were partially purified from ascitic fluids by ammonium sulfate precipitation (0 .33 g/ml), redissolved to the original volumes, dialyzed against phosphate-buffered saline (pH 7.2) and stored at - 20°C . Both crudearches and ammonium sulfate-purified preparations were titrated by EIA as described . MAhs typing Irotyping of MAba was performed by gel immunodiffusion (Oucx~rettt.orw and NltssoN, 1978) using supernatants from hybridoma cultures and a typing kit with subclass-specific antisera (ICN Immunobiologicals, Lisle, IL, U.S .A .). Western blotting Samples were separated by 12íe SDS-PAGE (Lwenn~tL~, 1970) or by 12ßßo urea-PAGE (Ttewue et al., 1971). Electrophoretic transfers to 0.43 pm nitrocellulose (Schleicher 8c Schuell, Keerre, NH, U.S.A.) were performed according to the methods of Towa~N et al. (1979) using 2 hr at 700 mA for urea-gels or 6 hr at 70 mA for SDS-

Monoclonal Antibodies to Myotoxin

677

gels . Blotted proteins were visualized according to the non-denaturing staining procedure of SYU and KAftAN (1987) and marked by punching pinholes . Residual blocking of nitrocellulose with 1N~ bovine serum albumin (BSA) in 0.02 M Tris, 0.5 M NaCI, pH 7.5 (TBS) for 2 hr at room temperature, was followed by MAbs diluted 1 : 100 in the homologous culture supernatants for 2-3 hr . Washing (3 x 10 min) was done with O.1Sk HSA-TBS, and then an affinity-purified goat-anti-mouse IgG-HRP conjugate (1 hg/ml in 0.19e BSA-TBS) was added for 2 hr . After washing, color was developed with 4-C1-1-naphthol and H,Oz according to Bio-Rad technical bulletin No . 1141 (Bio-Rad Laboratories, Richmond, CA, U.S .A.) . Immurtodjjjusion Ability of MAbs to precipitate myotoxin was tested by immunodiffusion in 1% agarose-phosphate-buffered saline plates (Oucxretet.otvY and Nttssotv, 1978). Samples (20 Kl) of myotoxin (0 .25 mg/ml) or B. riper venom (0 .5 mg/ml) were allowed to diffuse against an equal volume of MAbs (individually, in pairs, triplets, or as a pool) for 24 hr at 4°C. Plates were either photographed directly or washed with phosphate-buffered saline solution and stained with Amido Black IOB. HPLC purjflcatlon

and biotinylation of MAbs Ammonium sulfate-purified MAbs of the IgG, isotype were subjected to further purification by high performance liquid chromatography (HPLC) on an ABx ion~cchange column (Baker Chemical Co ., Phillipsburg, NJ, U.S .A .). Samples (3 ml) were dialyzed against 0.01 M KH zPO pH 6.0, and applied to the column. Elution was carried out with a linear gradient from 0.01 M KH,PO pH 6.0 to 0.25 M KH,PO,, pH 6.8 . Antibody activity of the peak corresponding to MAb was confirmed by EIA, as described. MAbs was concentrated by ammonium sulfate precipitation and dialyzed against 0.1 M NaHCO,, 0.15 M NaCI . Biotinylation was performed using N-hydroxysuccinimido-biotin (Sigma Chemical Co ., St . Louis, MO, USA) according to the method of STXftLt et al. (1983) . Unbound biotin was removed by dialysis against TBS . Competition binding assay Ability of different unlabeled MAbs to inhibit the simultaneous binding of biotinylated MAbs was tested by competition solid-phase antibody-binding assay according to the method of STXttLt et ol. (1983) with slight modifications. EIA-saturation curves for unlabeled MAbs were performed by incubation of varying concentrations of MAbs in myotozin-coated plates, and subsequent detection as described. Saturation curves for biotinylated MAbs were performed using an avidin-alkaline phosphatase conjugate (Sigma Chemical Co., St . Louis, MO, U.S .A .; 200 ng/ml) . After selecting the appropriate dilutiona, the competition binding assay was performed in triplicau wells as follows: 100 pl/well of an excess of unlabeled MAb (competing MAb) were incubated overnight at 4°C. Then, without washing, 50 ~l/well of biotinylated-MAb were added for 45 min at room temperature . After washing, binding of labeled MAb was detected with the avidin conjugate (200 ng/ml, 100 pl/well, 45 min at room temperature) . Results were expressed as percentage binding of labeled MAb, calculated as the ratio of net readings of labeled antibody in the presence of competing antibody, to net readings of labeled antibody in the ábsence of competing antibody, multiplied by 100. The experiment included controls to demonstrate self-inhibition of MAbs . 7tivo~rite ertryme~immunoassay A sandwich EIA was utilized to test the ability of pairs of MAbs to bind simultaneously to myotoxin in solution . EIA plates were coated with unlabeled MAbs (1 : 200 to 1 : 400) as described. Then, myotoxin was added (0.4 pg/100 ~l TBS-O.SaPo BSA/well) for 4 hr at room temperature . Plates were washed, and second MAbs (biotinylated) were added for 4 hr (100 pl/well of 1 : 250 to 1 : 1000 dilutions in buffer H with 0.590 BSA) . Labeled MAbs were detected with the avidin-alkaline phosphatase conjugate (250 ng/ml, 100 pl/well, 1 hr) and 4-methylumbelliferyl phosphate. Readings of controls in which myotoxin was omitted, were subtracted from readings of teat wells. All tests were run in triplicate . Using this technique, two MAbs were selected for developing an EIA for myotoxin detection. Different 'capturing' MAb concentrations for coating were tested by running calibration curves with myotoxin standards. Incubation times for antigen and biotinylated MAb were reduced to 2 hr . The detection limit of thin type of EIA was determined . Craxs-rnrctivity of MAbs with basic venom jroctions In order to investigate reaction of MAbs to B. aspen basic venom components, peaks 4, 5, 6 and 7 from the CM-Scphadex fractionation step were collected, lyophilized, and then each fraction was applied to a Stphadex G-75 column (1 .5 x 75 cm). Eluted fractions were pooled, dialyzed, and lyophilized. Binding patterns of MAbs to these fractions were tested by EIA. Fractions were analyzed by urea-PAGE. In order to obtain more refined resolution of the MAb-binding patterns to basic components of crude venom, individual tube fractions (3 .9 ml) obtained from the CM-Sephadex column were utilized to coat EIA plates . Binding patterns were tested similarly and wmpared to the protein elution profile. Negative ascites fluid was utilized an a control. All fractions were analyzed by urea-PAGE .

678

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Cross-rcactlons of MAbs with other vennms Cross-reactivity of MAbs towards other venoms was tested by EIA. Crude venoms (B. riper (Atlantic), B. srhlegelli, B. godmani, B, numrnjfer and B. picadoi; Instituto Clodomiro Picado) were coated on microplates at 4 and 2 Ng/well. Myotoxin (0 .4 Ng/well) was included as a control. These venoms were selected sins previous studies indicated cross-reactivity with anti-B. riper myotoxin serum (LOMONI'H et al., 1987b) . MAbs were diluted 1 : 100 and incubatedovernight (100 ~l/well) . Normal rabbit serum and mouse control ascites fluid were used as negative controls, whereas rabbit polyclonal scrum to myotoxin served as a positive control. The test was performed in duplicate. .~jfrnity chromatography isolation of myotoxin An immunoafflnity column was prepared by coupling MAbs to Affigel-10 according to the manufacturer's instructions (Bio Rad Laboratories). A partially purified preparation of myotoxin (peak 5 from the CMSephadex fractionation; 1.4 mg in 1 ml TBS) was applied to the column equilibrated in the same buffer, and after the absorbance at 280 nm returned to the baseline, elution was carried out with 0.1 M glycine, pH 3.0 . The eluate was immediately neutralized with 0.5 M Tris, pH 8.8 buffer . Protein obtained was dialyzed against water and lyophilized. It was analyzed by urea-PAGE and SDS-PAGE, and tested for myotoxic activity as previously described (LoMOwre et al., 1987a) . Neutralization of myotaxic activity Ability of ammonium sulfate-purified MAbs to neutralize CM-Sephadex-purified myotoxin was tested by preincubation experiments as described by LoMOxre et al . (1987a). An initial screening of individual and pooled MAbs was performed in 18-23 g mice (n = 2), at a ratio of S ~l MAb/pg toxin and a challenge dose of 20 pg. As a neutralizing control, horse antivenom was utilized . Absolute concentrations of each MAb were not determined, aad were assumed to be similar. Plasma creative kinase (CK; EC 2.7 .3 .2) values at 3 hr were used as an indicator of myonecrosis . MAbs showing a reduction in CK levels (as wmpared to mice injected i.m . with myotoxin alone) were retested in groups of three mice, at a ratio of 4 pl MAb/Ng toxin and a challenge done of 40 pg . A non-neutralizing MAb was included as a control. Significance of the differences from myotoxinigjected controls was estimated by Student's t-test. In addition to the measurement of plasma CK activity, muscle samples were taken, fixed in formalin, embedded in paraffin, and stained with hematoxylin-eosin for histological evaluation . RESULTS

Isolation of myotoxin Analysis of B. riper venom of urea-PAGE showed four fast-migrating bands corresponding to the most basic components (Fig . lA, lanes 3 and 4). Of these four bands, the uppermost was consistently observed to be less intense, whereas the remaining three were intense and similar to each other. The CM-Sephadex elution profile of B. aseer venom was essentially the same described by GUT1Z$RREZ et al. (1984) with the exception of peak 8, which was absent . The purified preparation of myotoxin is shown in Fig. lA, lanes 1 and 2, as a single band by Coomassie Blue R-250 staining. Production of anti-myotoxin hybridomas and MAbs Approximately 700 hybridomas were generated from the fusion, of which eight showed production of myotoxin-specific antibody, by EIA. Proportionally, mouse blood cells feeders yielded about twice the number of hybrids as did sheep feeders. Cloned cell lines were established and referred to by the numbers 1- 8. Clone 7, originally typed as IgG3, stopped producing antibody . All the remaining clones produce IgG, with the exception of clone S, which produces IgM. Titration of crude ascites and ammonium sulphate-purified MAbs by EIA on myotoxin-coated plates showed similar results. MAbs-2, 3, and 4 showed higher titers (> 1 :10 as compared to MAbs-1, 5, 6 and 8 (about 1 :10'). Western blotting Specificity of MAbs was initially evaluated by Western blotting. No reactions were obtained on blots from SDS-PAGE, except for MAb-3 which gave a very faint band at 16,000 mol. wt. Blots prepared from urea-PAGE showed reactions of variable intensities

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679

FIa . 1 . LTRPA-PAßE ANALYSIS OF MYOTOXIN AND VL~IOMS. 1 and 2 : myotoltin, purified by CM-Srphadex recycling ; 3 and 4 : B. aspor (Atlantic type) venom ; 5 : B. srhlegdü; 6 : B. picadoi; 7 : B. godmani: 8 : B. nummjfn (Pacific type) . (B) Immunoaffmity chromatography using MAb-4 immobilized on Affígel . 1 : starting material, peak S from CM~ephadex; 2 : B. aspor venom ; 3 : myotoxin, eluted at pH 3 .0 ; 4: unbound material . (A)

corresponding to the group of fast-migrating bands of B. riper venom (Fig. 3). Exact location of reactions was greatly facilitated by the use of mild pre-staining of protein bands and pinhole marking of the nitrocellulose paper. MAbs showed cross-reactions between the four bands, although some differences in the reactivity patterns were observed . Reaction of MAb-1 was very faint, but clearly visible on the original blot. Increasing antibody concentrations did not improve detection . MAbs-1, 6, and 8 tended to react stronger against the lower band, whereas MAb-5 reacted slightly better with the middle band corresponding to myotoxin (Fig. 3). Reactions of MAbs-3 and 4 were the strongest, however MAb-3 reacted with the lower band whereas MAb-4 did not (Fig. 3). MAb-2 reacted similarly with all four bands. The experiment was repeated with identical results. Immunod~(fusion

A pool of equal parts of each MAb formed a sharp preeipitin line against myotoxin and venom (Fig. 4A). A reaction of identity was observed between rabbit anti-myotoxin serum and the MAbs pool in their reaction against myotoxin (Fig. 4B). By testing each MAb individually, it was found that either MAb-3 or MAb-4 were able to precipitate myotoxin (Fig. 4C). Results were confirmed by recloning one of these hybridomas (MAb-3) and repeating the immunodiffusion test. In some experiments, a very faint spur towards MAb3 was detected (Fig. 4G). The remaining non-precipitating MAbs were tested similarly in

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FIO . Z . EI,EGTROPHORETIC ANALYSES OF BASIC VENOM FRACTIONS .

Urea-PAGE . 1 : fraction S, after a second CM-Sephadex cycle; 2 and 9: B. riper venom; 3 : fraction 7b ; 4: fraction 7a ; S: fraction 6a ; 6: fraction Sb ; 7: fraction Sa ; 8:fraction 4a . (b) SDSPAGE . 1 and 9: mol. wt markers (x 10-j; 2: fraction 7b ; 3: fraction 7a ; 4: fraction 6a ; S: fraction Sb ; 6: fraction Sa; 7: fraction 4a ; 8: fraction S, after a second CMSephadez cycle; 10 : B. riper venom. All samples were reduced with 2-mercaptoethanol. (a)

pairs or triplets against myotoxin, but no precipitation lines were detected . Presence of dimers in the myotozin preparation was investigated by SDS - PAGE under reducing and non-reducing conditions . In addition to the main 16,000 mol. wt band, an additional band

Monoclonal Antibodies to Myotoxin

68 1

F~a. 3 . wssre~uv swTTVVas of Mor~oci.oruz. wxr®oniss wawnvsr n'voroxnv wrm B. aspor veNOM sepwawxßD sv uasw-PAGE. 1- 8: MAbs 1- 8 ; N : negative ascitea control; S : non-denaturing staining of proteins on nitrocellulose; V: B. asps venom; M : myotoxin . The arrow head in MAb-S points to a faint reaction with a slower-migrating protein in crude venom. After mild stain of blots with Amido Black 10 B, exact positions of bands were marked with pinholes . Reaction of MAb-1 was very faint, but clearly visible on the original blot .

at about 22,000 was observed in the unreduced sample . After reduction with 2-merc~ptoethanol, a single band of 16,000 mol. wt was obtained . Competition SABA and two~ite EIA

Results of competition binding assay are summarized in Table 1 . Some MAb combinations showed clear competition or non~ompetition (percentage binding values approaching 0 or 100, respectively), whereas others resulted in partial competition values . Table 2 summarizes results of the two-site EIA using combinations of MAbs. Myotoxin was efficiently bound by several pairs of MAbs. In the case of MAbs-3 and 4, myotoxin could form a "sandwich" between two molecules of a single MAb (Table 2). Using MAb4 for coating and biotinylated MAb-3 for developing, calibration curves in the range of 400 to 6 ng myotoxin were performed (Fig. Sa). Fluorescence readings were proportional to the log of myotoxin added to the wells, and to the coating concentration of capturing MAb (Fig. SA). The detection limit of this type of EIA (using a 1 : S00 dilution of MAb-4 for coating) was approximately 1 .5 ng (Fig. SB). Crass-reactivity of basic venom fractions:

Peaks 4, S, 6 and 7 from the CM-Sephadex column were further fractionated on a G-73 column . Fractions 4 and 6 eluted as single peaks, whereas fractions S and 7 generated two peaks each. These new fractions were designated 4a, Sa, Sb, 6a, 7a and 7b. SDS-PAGE

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B. LOMONTE and L. I{AHAN

FiO . 4. OBL INDNUNOD~FSISION. 1 : MAba pool ; 2: myotoxin; 3: B. aspor venom; 4: rabbit anti-myotoxin serum. (B) 1: myotoxin ; 2: MAbs pool ; 3: rabbit anti-myotoxin serum; 4: B. aspor venom. (C) m: myotoxin ; 1- 6: MAbs 1- 6. (A)

analysis is shown in Fig. 2b . All fractions migrated as single bands of approximately 16,000 mol. wt after reduction . Urea-PAGE analysis (Fig. 2a) showed that mobility of fractions correlated with the elution order in CM-Sephadex, and that there were no differences between the two peaks resolved by gel filtration of fractions 5 and 7 (i.e. fractions Sa and Sb ; or 7a and 7b). These fractions were coated onto EIA plates and binding of MAbs was tested (Fig. 6). Readings using fractions SA and SB, or 7a and 7b, were essentially the same, and for simplicity only one of each is presented in Fig. 6. MAbs1, 6 and 8 showed a similar pattern, reacting slightly stronger towards the most basic fraction. Clear differences were observed among MAbs-2, 3, 4, and 5 (Fig . 6). The experiment was repeated using randomized position of the fractions on the EIA plates, with identical results.

683

Monoclonal Antibodies to Myotoxin TAeLe 1 . COMPETITIVE HINDING OF MAbs To B . riper MvoTOxIN

Competing MAb (unlabeled)

Labeled MAb

1

2

3

4

S

6

8

1 2 3 4 6 8

6t2" 14t1 86t6 32t3 19t1 9t1

4t2 9t2 88t8 38t3 22t4 lOtl

103f14 24t2 7t1 29t6 22t3 38t4

lSt3 lOt2 89t6 13t3 9t1 14f1

36t1 17t1 63t1 6t4 4t4 33t1

lOtl 2t2 72t7 14t3 24t2 8t4

4t2 lOt2 lOlt13 33t9 33t3 3t1

" Percentage binding of labeled MAb = 100 x [net fluorescence of labeled MAb in the presence of competing MAb/net fluorescence of labeled MAb in the absence of competing MAb], as described in Materials and Methods . Each value is the average of three determinations t 1 S.D.

Twst.E 2. Two-stTE EIA USING PAIRS OP MAbs To B. riper MvoTOxtN

First MAb 1 2 3 4 3 6 8

Fluorescence (arbitrary units) " Secon d MAb 1 21 t 26 f 117 t 209 t 19 t 50 t 21 t

2 1 2 4 11 1 3 1

78 t 1 179 t 1 943 t 38 1632 t 47 236 t 7 280 f 2 237 t 5

3 3392 t 5016 t 5272 t 6182 t 5310 t 6242 t 3903 t

4 163 276 197 299 249 197 132

1473 1849 2873 2968 2413 2886 2661

t t f t t t t

6 29 SO 37 88 13 84 106

343 318 1627 2438 632 982 1078

t t t t t t t

8 12 50 118 224 92 92 77

-26 -47 268 416 - 30 C - 20

t2 t1 t3 t9 t 1 t1 t1

" Average of three determinations t 1 S .D . Readings of antigen-containing wells were corrected by subtracting readings from controls without antigen . EIA plates were coated with first MAb, then myotoxin was added, followed by biotinylated MAb (second MAb) and avidin-alkaline phosphatase conjugate, as described in Materials and Methods .

Since the electrophoretic composition of the pooled fractions was not homogeneous (Fig. 2a), binding patterns of MAbs to basic components were further investigated by performing EIA on samples from individual tubes eluting from CM-Sephadex. Figure 7 shows the protein elution profile corresponding to peaks 4-7 and the reactivity of each MAb towards each fraction . Results were confirmed by repeating the CM-Sephadex fractionation and EIA. Urea-PAGE was performed on all tubes, giving a pattern consistent to that shown in Fig. 2a. Cross-reactions of MAbs with other venoms Rabbit anti-myotoxin serum control strongly cross-reacted with the different crude venoms tested (Table 3). Only MAbs-2, S and 6 showed a significant (~ 3 times control values) cross-reactivity towards B. nummifer venom (Table 3). Urea-PAGE of the crude venoms, compared to B. riper venom, is shown in Fig. lA. Immunogffinity isolation of myotoxin Protein obtained from the MAb-4-Affigel column is shown in Fig. 1B, lane 3. By SDSPAGE, a single band (16,000) appeared under reducing conditions (not shown) . Injection ofapproximately SO Fig of toxin i.m. into two mice induced a ten-fold increase in CK levels at 3 hr, compared to mice injected with phosphate-buffered saline .

684

B. LOMONTE and L . KAHAN

s

rA

4

2

I

8 6

I

I

12

25

I

I

50

I

1

100 200 400

B

4

2

L_a__, --"' I

0.1

0.2

. p.4

0.8

1 .8

1

3

I

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8

12

25

Log ng myotoxin

I

50

FIG . S . T~VO-SITE EIA FOR MYOTOXIN DSfEGTION USING MONOCCANAL AN'17HODIES 3 AND 4 . (A) plates were coated with different dilutiona of MAb - 4: (D) 1 : 250; ( " ) 1 : 500; (V)1 : 1000; (p) 1 : 2000. Then, myotoxin was added, and detected with biotinylated MAb-3 and avidin-alkaline phosphatase conjugate, as described in Materials and Methods . AU : arbitrary units. ($) determination of the detection limit using MAb~ for coating at 1 :500 dilution .

Neutralization of myotoxicity MAbs-1, 2, 6 and 8, did not reduce CK levels at a proportion of S ~l MAb/pg toxin (data not shown) ; MAbs-3, 4, S, a pool of equal parts of each MAb, and horse antivenom reduced CK to levels comparable to those of mice injected with phosphate-buffered saline . Neutralization by MAbs-3,4, and 5 at a ratio of 4 pl MAb/~,ig toxin is shown in Fig. 8 . Histological evaluation of muscle sections correlated with results obtained by CK quantitation. DISCUSSION

Seven murine hybridoma lines secreting MAbs to B. riper myotoxin have been established . Their specificities have been characterized by Western blotting and EIA. Reactivity patterns indicate the presence of at least four proteins recognized by MAbs in crude venom . These proteins have a common subunit mol. wt (about 16,000 by SDSPAGE) but slight differences in charge, observed both by Western blotting and cationexchange chromatography followed by EIA. These findings suggest that the crossreacting components may be variants or isoforms of myotoxin. However, information on biological, enzymatic, and chemical characteristics of each purified protein would be necessary to confirm this . A number of recent studies describe the presence of multiple toxin forms in snake venoms (HAZLETT and DENNIS, 1985 ; RAEL et al., 1986 ; Du 130URDIEU et al ., 1987 ;

Monoclonal Antibodies to MyotoIrill

68 3

0

x

a

0

F1ß. 6. BII~IDINO OF MONOCLONAL ANrIHODIES TO POOLED BASIC VENOM FRACTIONS BY EIA . AU : arbitrary units; M: myototin ; V : B, riper venom; 4-7: fractions 4a, Sa, 6a, and 7a, respectively; a: MAb-1 ; b: MAb-2; c: MAb-3; d: MAb-4; e: MAb-S; f: MAb-6; g: MAb-8; h : negative ascites control. EIA plates were coated with venom fractions and binding of MAbs was detected with an anti-mouse conjugate as described in Materials and Methods.

FAU1zE and BoN, 1987 ; Jol~vsox and BIEBEIt, 1987). Thus, present findings with B. riper myotoxin are not surprising . Since the venom used in this study is a pool from many specimens (more than SO), it would be of interest to investigate if cross-reacting components correspond to allotypic variants in the population, or isotypic variants present in venom samples from individual specimens. This type of analysis for crotoxin isoforms in Crotales durissus tutus has shown that venom variability can be explained to a great extent by the complexity of venom obtained from individual snakes (FAU1zE and BoN, 1987). Titration curves of the different MAbs, as well as intensity of reactions in EIA and Western blottings, all indicate a higher affinity for myotoxin of MAbs-2, 3, and 4, as compared to MAbs-1, 5, 6, and 8. MAbs showed interesting differences in their reactivity patterns against myotoxin-related components . Results obtained by Western blotting correlate well with those obtained by 13IA binding studies (Figs 3, 6, and 7) . MAbs-1, 6 and 8 had specificity patterns very similar to each other, suggesting that all three might be directed against the same epitope or at least very close epitopes . Indeed, by competition binding assay, reciprocal inhibitions ranging from 67 to 96% were obtained between MAbs-1, 6 and 8. On the other hand, MAbs-3 and 4 probably recognize different epitopes, since their binding patterns differ strikingly regarding reactivity to peak 7. In support of this, results showed that binding of an excess of MAb-4 to myotoxin did not interfere with binding of biotinylated MAb 3 . However, MAb-3 inhibited substantially

B. LOMONTE and L. KAHAN

_o X

Q

c ó

Fraction number FIG. 7 . REFINED BINDING PATTERNS OF MONOCLONAL ANTIBODIES TO BASIC VENOM FRACTIONS HY EIA. (p) Absorbance at 280 nm ; ( " ) Fuoesoence; AU: arbitrary units; a: MAb-2; b: MAb-3; c: MAb4 ; d: MAb-5; e: MAb-6; f: MAb-8. Pattern of MAb-I (not shown) was identical to that of MAb-6. Negative ascites control value were subtracted from all readings . Samples from individual tubes eluted from the CM-Sepbadex column were coated on EIA plates, and binding of MAbs was detected with an anti-mouse conjugate as described in Materials and Methods.

TABLE

3.

CROSS-REACI1VrrY OF

MAb3

TO B . aspen MYOTOXIN WITH CRUDE VENOMS OF OTHER SPECIES BY

EIA.

Fluorescence (arbitrary units)' Antibody 1 2 3 4 5 6 8 NA RP NR Myotoxin 604 2208 3976 3498 1345 1458 548 133 3994 146 B. godmani 221 179 181 121 213 111 66 113 2462 105 B. schlegelü 200 276 274 89 314 159 70 124 2133 99 B. numm((er 160 2278 l53 91 725 384 63 127 3367 92 B. picadoi 203 183 193 121 245 Ï48 75 105 1433 88 B. aspor 726 3507 2812 632 1372 715 169 2529 2l4 3987 'Average of two determinations, as described in Materials and Methods. Underlining indicates positive reactions, defined as > 3 times than negative control readings . NA : negative ascites control; RP : rabbit polyclonal antiserum to B. aspen myotoxin ; NR : normal rabbit serum. Antigen

binding of MAb-4. Non-reciprocal inhibitions can be seen in this type of assay, and may reflect a variety of complex phenomena, such as epitope stability and allosteric changes in antigen conformation induced by the binding of one of the MAbs (LANE et al., 1985). The

Monoclonal Antibodies to Myotoxin

687

240

É 3 z .> ]L U

180

tso 60 0

U_ mt

FiO. S . NEUTRALIZATION OF MYOTOXIC ACTIVITY OF PURIFIED MYOTOXIN HY MAbs-3, 4 AND S . Bars represent mean f S.D. of three determinations in mice. Statistically significant (p < 0.05) differences from myotoxin-injected mice are indicated by (" ); nit: myotoxin; pbs: phosphato buffered saline control; MAba are indicated by their number . CK : creatine kinase. One CK unit of activity is defined as the phosphorylation of one nanomole of creative per min at 25°C .

overall evaluation of Western blottings, EIA binding patterns and competition binding experiments suggest that at least four epitopes are being recognized by this set of MAbs. The observed differences in MAb reactivity patterns towards the myotoxin-related components might be exploited in several ways, such as the immunoaffinity separation of components, and neutralization studies, which could help to elucidate their structure - function relationships . One example of such possible applications was the use of MAb-4 in the one-step separation of the two bands present in CM-Sephadex peak S by immunoaffinity chromatography (Fig. 1B) . The binding pattern of this MAb strongly supported its use, since it had the lowest reactivity towards peak 4 components, while having a high affinity for peak S components . Gel immunodiffusion experiments showed precipitation of myotoxin by either MAb-3 or MAb~. This implies that the epitope recognized is repeatedly available, with a minimum number of two . Results obtainod by two-site ElA also demonstrate the simultaneous availability of at least two identical epitopes (recognized by MAbs-3 or 4) on myotoxin in solution. Formation of antigen dieters (or larger aggregates) would explain this observation . Alternatively, the epitope(s) might be repeated in the monomer subunit . Aggregation of subunits into dieters in venom phospholipases has been reported (ISHIMARU et al., 1980; MARAGONORE et al ., 1984; WELC[->ES et al ., 1985; RENETSEDEIt et al., 1985) and can be concentration-dependent (DEEMS and DENNIS, 1981) . In the present case, it was found that a small but detectable proportion of myotoxin migrates as a band of about 22,000 mol . wt by SDS-PAGE (unreduced), indicating some degree of dinier formation . Results of gel filtration chromatography of peak S also support this concept, since the two peaks resolved on Sephadex G-75 had the same electrophoretic composition . Thus, dieter formation probably accounts for the ability of single MAbs to form an efficient `sandwich' by two-site EIA or precipitate myotoxin in gel immunodiffusion . By two-site EIA, several pairs of MAbs were able to bind myotoxin in solution. Results indicated MAb-4 as the best `capturing' antibody, and MAb-3 as the best `detector'. Using this pair of MAbs, an ElA for myotoxin detection was designed . The use of biotinylated second MAb and avidin-alkaline phosphatase conjugate resulted in low background and high sensitivity, as described (TIJSSEN, 1985). This ElA clearly detected

688

B . LOMONTE and L . KAHAN

myotoxin at the nanogram level, and can be performed in a few hours . It may find application for a variety of purposes . Despite the clear cross-reactivity of polyclonal anti-B. riper myotoxin antibodies towards other snake venoms, only three of the MAbs obtained showed cross-reactivity and all three reacted with the venom of B. nummifer . A myotoxin with mol . wt of 16,000 (by SDS-PAGE), able to form dieters, and devoid of phospholipase A2 activity, has been isolated from this venom, and cross-reacts with anti-B. riper myotoxin polyclonal serum with a pattern of partial identity (GUTn$RREZ et al., 1986a) . It is likely that cross-reactivity of MAbs towards crude B. numm(fer venom is due to its myotoxin, although this would have to be confirmed . Of the seven MAbs generated, three independently showed neutralizing ability towards myotoxic activity of purified myotoxin . Neutralization of phospholipase and other activities of myotoxin by these antibodies remains to be investigated . Acknowledgements - We thank Drs Wwrr-Jtt SYU, J . M . Gtrri$axaz and E . Moat~o for helpful discussions, and Ms CwxoL Sttvwuco and Hanow Goooutwty for expert technical assistance . This work was carriod out at the University of Wisconsin Hybridoma Facility and Department of Physiological Chemistry, and supported in part by a Ftiilbright Research Scholarship to B . L . (grant # 08387) . REFERENCES Bore, M . A ., Lesst.nv, B. A ., ODtau., G . V . and OwtvaY, C . L . (1987) Affinity purification of antibody to myotoxin a from Crotales viridtr viridis venom . Toxicon 25, 133 . Boi.wFtos, R. (1982) Las serpientes venenosas de Centroamérica y el problems dal ofidismo . Primers parte: aspectos zoológicos, epidemiológicos y biomédioos . Rev . Cost. Cienc. Mdd. 3, 163 . Ci~mvzm, H . (1979) Fractionation of myonecrotic factor isolated from the venom of baba (?Yimeresurus flavovirldLs) . Jpn . J. mad. Sci. Biol. 32, 117 . Da BLA3, A . L., Rw~rtvwrrwaxta, M. V. and Mostntwtvtv, J`. E . (1983) Estimation of the number of monoclonal hybridomes in a cell-fusion experiment . Methods Enzymol . 92, 36 . Dots, R . A . and Detvrns, E . A. (1981) Phospholipase At from cobra venom (Ngja ngja ngja) . Methods Enzymol. 71, 703 . Du BouxDistt, D . J ., Kwwwauctu, H. and Stimte, W . T. (1987) Molecular weight variations in the diversity of phospholipaae A, forms in reptile venoms. Ta:ricon 25, 333 . FAHIANO, R . J . and Tu, A. T . (1981) Purification and biochemical study of viriditoxin, tissue damaging toxin, from prairie rattlesnake venom . Biochemistry 20, 21 . Fwutes, G . and Botv, C . (1987) Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake (Crotalua dtuissus terrificus . Toxicon 25, 229 . ßuTlEwtez, J . M ., Owtvav, C . L . and ODt:Lt G . V . (1984) Isolation of a myotoxin from Bothroptr riper venom : partial charactervation and action on skeletal muscle . Toxicon 22, 115 . Gu~nEaxaz, J . M ., LOMONI'E, B . and C$aD~s, L . (19ß6a) Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummjjer. Taxiton 24, 885 . Gvr~texEZ, J . M ., LoMOxre, B ., Cttwves, F ., Moat:tvo, E . and Crno~s, L . (19ß6b) Pharmacological activities of a toxic phospholipase A isolated from the venom of Bothrops riper. Comp. Biochem. Physiol. 64C, 139 . Hwteats, J . B . and MwcDotvet,t., C . A . (1981) Phospholipase A, activity of notexin and its role in muscle damage . Taxiton 19, 419 . Hwzt.err, T . H . and DeNtvts, E . A . (1985) Affinity chromatography of phospholipase A= from Ngja ngja ngja (Indian cobra) venom . Toxicon 23, 457 . Istßtrtwnu, K ., Kutwaw, H. and OFnvo, M. (1980) Purification and properties of phospholipase A from venom of 7Yimeresurus Jlavoviridis (Habu snake) . J. Biochem. E8, 443 . Jorttrsotv, G . R. and Btt~x, A. L . (1987) Mojave toxin : multiple forms in crude venoms, purified and recombined toxin . Toxfcon, 25, 145 . KOxLex, G: and Mtisrettv, C . (1976) Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion . Ear. J. Immunol. 6, 511 . i.wr~n.r , iJ . K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Natwr 227, 680 . LAtvE, D . P ., Gwtvreotv, J ., BttEtvtvwtv, S . P . J . and MoL$, S. E . (1985) Investigating the specificity of monoclonal antibodies to protein antigens using ß-galactosidase fusion proteins . In : Investigations and Exploitation of Antibody Combining Sites, p . 75 (REID, E ., Coox, G. M . W . and Moteaé.; D . J ., Eds). New York : Plenum Freas .

Monoclonal Antibodies to Myotoxin

68 9

Lontotlt~, B., Otmáxaaz, J . M. aad Mwrw, E. (1985) Isolation from a polyvalent antivenom of antibodies to a myoto~n in Bothrop~s sapa snake venom. Taxlcon 23, 807. LoMOtaTe, B., Otmt3xaez, J. M., MoaErto, E. and Ctoto~s, L. (1987x) Antibody neutralvation of a myotoxin from the venom of lßothrops saper (terciopelo). Toxieon 25, 443. Lot~orrt8, B., Mottwo, E. and Otrrtt3aaez, J. M. (1987b) Dotation of proteins antigenicauy-related to Bothrop~s ~spar myotoxin in crotaline snare venons . Toxkron 1S, 947. Mwnwaotvont:, J. M., Mt :attTaw, O., Ceo, W., WtsLCtms, W., I{áznv, F. J. and Heinrikson, R. L. (1984) A new class of phospholipasea A, with lysine in play of aapartate 49 . Functional consequences for caldum and substrate binding. J. biol. Clam . 259, 13839. Mtms, D. (1986) Myoto~c activity of phoapholipeaea A= isolated from cobra venons : Neutralization by polyvalent antivenom. Toxirnn 24, 1001 . Mra4, D. end Swt~rtntw, Y. (1986) Isolation and characterization of myotoac phospholipases A, from crotalid venons . Toxirnx 24, 161 . Muas, D., EttatavFeLO, M. and $AMFJIMw, Y. (1983) Local narotizing effect of snake venons on skin and muscle : Relationship to serum creative kinase . Toxirnn 21, 393. Ouctrrgawrnr, O. and Nttssotv, L. A. (1978) Immunodiffusion and immunoelatrophoreais . In : Handbook of Experimental Immunology, Vol. 1, Immunochemistry, p. 19 (Wt3ne, D. M., Ed .) . Oxford : Blackwell Scientific PubGcationa. OwrtaY, C. L., CAMERON, D. and Tu, A. T. (1976) Isolationof myotoxic component from rattlesnake (Crotales vuldis vuldis) venom. Electron microscopic analysis of muscle damage . Am . J. Path . i5, 149. OwNaY, C. L., ODELL, G. V., Woons, W. M. and Cot.nEaa, T. R. (1983) Ability of antiserum to myototdn a from Prairie rattlesnake (Crotahas viridis vuldis) venom to neutralize local myotoxidty and lethal effects of myotoxin a and homologous crude venom. Taxicon 21, 33 . OwxaY, C. L., Co~aEaa, T. R. and Onat .t ., O. V. (1986) In vivo ability of antimyoto:dna serum plus polyvalent (Crotalidae) antivmom to neutralize Prairie rattlesnake (Crotahis viridis virldis) venom. Taxirnn 24, 197. RAEL, E. D., SwLO, R. J. and ZEpEnw, H. (1986) Monoclonal antibodies to Mojave t07[in and use for isolation of cross-reacting proteins in Crotah~.s venons . Toxirnn 24, 661 . Rwsrsenaiz, R., Bxtnvm, S., Dtncsraw, B. W., DaP.nrrtt, J. and StOLEa, P. B. (1985) A comparison of the crystal structures of phospholipasea A, from bovine pancreas and Crotales atrox venom. J. biol. Chem. 260, 11627. ST~ittz.t, C., MtaatwNO, J., S~roctcEn, J., S`rwtstn3t.tN, Th ., Hwxtrra, P. and Twtu(cx, B. (1983) Distinction of epitopes by monoclonal antibodies . Methods Ertrymol . 92 . 242. SYU, W.-J. and ICwttwrt, L. (1987) Use of protein-stained immunoblots for unequivocal identiScation of antibody apecifidties . J. immunol. Methods (in press) . TttssEN, P. (1985) Prnctice and Theory ojEnryme Immuxoassays. Amsterdam: Elsovier Silence Publishers . TOWHtN, H., STwErtat.ttv, T. and GoAUOx, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : praadure and some applications. Proc. nota . Aced. Sci. USA 76, 4330. TxwuE, P., Mtzust~ttMw, S., Lowtev, C. V. and NoMttaw, M. (1971) Reconstitution of ribosomes from subribosomal components . Methods Enrymol. 20, 391 . WEL.atFS, W., FEtsttEte, D., Lwttosttutz, W. and MwnwaotvoxE, J . M. (1983) A rapid method for the purification of monomerkc and/or dimeric phoapholipxses A= in crotalid snake venons . Taxicox 23, 747.