Toslron, VoL 20, No. 3, pp. 563-569, 19ß2Prmoed in Gmat Brihm.

coal-0lol~zo~ow3-m sos.aorop 1982 Perpmm leer Ltd.

TITRATION OF ANTISERUM TO SOUTH AMERICANRATTLESNAKE (CROTALUS DURISSUS TERRIFICUS) VENOM

BY MEASURING INHIBITION OF PHOSPHOLIPASE A2

ACTIVITY

MARIA HELENA DA SILVA* and OTTO GUILHERME BIERWHO/PAHO Immunobgy Research and Training Center, Insdtuto Botenfan,

Sâo Paulo, SP, Brazil

(Acceptedfor publication 22 October 1981)

M. H . DA SavAand O.G. BmL Titration ofantiaerum to South American rattle®ake (Crotalesdivisorsterrificus) venom by inhibition of phospholipese AZ activity. Toxicon 20, 563-569, 1982.-Horseantiserum to the venom of Crotales derissus terrificus, a South American rattlesnake, inhibits thephosphofipase activity of the crude venom. There is a close relationship between this inhibitoryproperty and the neutralizing potency of the antiserum in viva. This may provide the basis for arigorous standardization of anticrotalic venom in vitro.

INTRODUCTION

TI->E vENOM of Crotalus durissus temficus, the South American rattlesnake, like other snakevenoms is a complex mixture of polypeptides and proteins with toxic and enzymaticproperties (MEBS,1970 ; ZLOTxIN,1973). Its main toxic component, crotoxin, was obtained in1938 by $LOTTA and FRAENKEL-CONRAT, as a crystalline protein having phospholipaseactivity. In fact crotoxin is a complex between a highly basic phospholipase AZ(phosphatidate 2-acylhydrolase EC 3.1 .1 .4) and an acidic polypeptide, crotapotin, whichpotentiates the toxicity but inhibits the enzymatic activity of the phopholipase (HAHIItMANNand Rt)ssA1~N, 1970 ; RÜBSAMEN et al., 1971 ; HENDON and FRAENxEL-CONRAT, 1971).

Crotoxin represents 65-68% (w/w) of the crude venom and shows an LDSO of about0.070mg/kg after i .p. inoculation in mice . It accounts for the high toxicity of the crudevenom : i .p . LDSO, 0.090mg/kg, in mice (HANASHIROet al., 1978). In the same species the i .v.~so of crotamine, another neurotoxic component present in venoms from specimenscollected in Southern Brazil and Argentina (GoxçALVES and VIEIRA, 1950 ; SCHENBERG,1959), is 4 mg/kg (HABERMANN and CIIElvc-RAUDE, 1975).

It has been previously shown that rabbit anti-Crotalus phospholipase Az neutralizes theenzymatic and neurotoxic activities of the purified enzyme as well as of crotoxin and crudevenom (HANASHIRO et al., 1978). This led us to investigate whether the inhibition ofphospholipase activity in vitro would parallel the neutralization of toxicity in viva .

+Fellow of CNPq, the Brazilian Council for Research Development and Technology.

563

564

MARIA HELENA DA SILVA and OTTO GUILHERME BIER

MATERIALS AND METHODS

VenomOne batch of crotatnine positive C. durissus terrificus venom was used throughout the experiments . It was

prepared at Butantan Institute (Säo Paulo, Brazil) by pooling venomfrom animals captured in the Southern regionof Brazilanddrying it overCaCh under normal pressure, at37°C. The driedvenomwasremoved fromthe desiecatorand kept in tightly closed vials of 4°C. Two samples of this batch (lO.Omg each), after exposure to the roomatmosphere for 24 hr, weretransferred to aseconddeaiocator and maintained over CaCh under normal pressure, atroom temperature, until constant weight. The samples were dissohrod in 0.15 M NaCI and used to establish astandard curve by plotting absorbencyat 280 nmvsvenomooneentration in mgper mL Theconcentration ofvenomsolutions used throughout the experimentswas obtained by reference to this curve. All venom solutions werekept at- 20°C until use.

Antiphospholipase A s serumAntiphospholipase A~ serum was prepared by injecting rabbits with purified Crotalus phospholipase A, isolated

from crotoxin, as dearibed previously (HANASHIRO et aL, 1978). The whole serum was lyophilized and dissolved indistilled water immediately before use.

Anticrotalic t+erwm seraSix diüerent batches of anticrotalic serum, as prepared at Butantan Institute for serumtherapy, were used . These

sera were prepared in horses according to a routine immunization procedure which involves s.c. injections ofaotamine positive venom emulsified in Freund's complete or incomplete adjuvant on two consecutive weeks,followed by booster injections of antigen without adjuvant. Sera showed potencies corresponding to theneutralization of 2.0-4 .5 mg of crude venom per ml of andvenom, when tested into pigeons . Sera were dialyzedagainst saline (0.15 M NaCI) to remove phenol and kept frozen at - 20°C. Corrections for dilution doting theprocess were made by precipitating proteins with ammonium sulfate at 50/ saturation, dissolvingthe precipitate in0.25 M acetic acid and determining the absorbency of the solution at 280nm.

Phospholipase A Z assayPhaspholipase A 2 activity in venom solutions or mixtures ofvenom plus antivenom, was determined by the egg

yolk-hemolysis test, as described by GRASSMAN and H~rnvtG (1954 with slight modifications : O.SOmI of a 6.4%(w/w) egg yolk emulsion in saline containing 0.02 sodium suds, was added to SOOpI of the test solution andincubated at 37°C. Aftera defined hydrolysis tithe (30thin-16 hr) 30pl samples ofthe hydrolyaates were transferredto 0.30 ail ofa standardized sheep red bloodcell suspension (4.5-5.0 x l0s cells/ml) plus 26 tail of saline. After 1 hrat 37°C, suspensions were centrifuged for 15 mirr at 400gand the absorbanciea ofthe supernatants read at 540nm.Lysis (/) was estimated by reference to a completely hemolyzod mixture obtained by adding 0.30 m1 of thestandardized cells to 26 ml of distilled water.The test solutionscontainingvenom plus antivenom wereprepared bymixing constant volumes ofvenom solution with variable vohtmes of antiaenrm and saline to the final volurrte of500W. In most experiments normal horse serum (30 pl) was added to the teat solutions. All test solutions (venomsolutions orvenom plus antivenom mixtures) werepre-incubated for 45min at 37°Cbefore addition of the egg yolkemulsion, in the same way as were the trrixturea prepared for the neutralization test in mice .

Neutralization test in mice and pigeonsMale Swiss mice of20-25 g were injected with 0.20ml ofvenom solution or venom plus antivenom rrtixtures per

10 g of body wei~t . The mixtures were pro-incubated for 45 thin at 37°C before being injected. Mortality wasrecorded up to 24 hr. r u se and its confidence limits (for 19/20 probability) were estimated by the method ofLrfct~p and wucoxoN(1949)on the basis of seven points on the regression line. Injections were given i.p. andsixanimals per done wero leafed.The neutralizing potency of horse antisera was determined by Vital Brazil's method by injecting pigeons of

250-350g with mixtures of 1.Om1 of undiluted serum plus LOmI ofvariable dilutions of crude venom (MLD Corpigeons is 0.015 mg). After30 min ofincubation at 37°C, mixtures were injected i.v . Deathswere recorded 24hr aater .

RESULTS

Phospholipase AZ activity ojthe whole venom. Effect ojnormal serum on phospholipase activityPhospholipase activity could not be detected up to 120 min with 27 pg of crude venom in

saline . Addition of 30Et1 of normal horse or rabbit serum to the test solution led to 100'/hemolysis within 90 min. This activation effect was not observed if the serum was previouslydialyzed (0% hemolysis after 120 min ofhydrolysis). In either case, l6 hr hydrolysates caused100'/ hemolysis.

Inhibition of phospholipase A Z activity by horse anticrotalic serumHorse hyperimmune serum raised against the whole venom inhibited its phospholipase

activity. In a typical experiment, 27 ug of venom were mixed with variable volumes ofantiserum diluted 1 :10. The phospholipase activity ofthe mixtures decreased as the volume ofantiserum increased . With SOpI of serum "E" (batch C-23-77-118, neutralizing potency inpigeons4.5 mg ofvenom per ml)only 10~ hymolysis wasdetected after 16 hr ofhydrolysis. Ata higher level of antiserum (70p1) no lysis was obtained (0'~ hemolysis~ Residualphospholipase activity of partially inhibited mixtures was enhanced by addition of normalserum For instance, the mixture containing 50~1 of serum "E~ caused 100'/ hymolysis when30p1 of normal serum were also added to the test solution . No enhancement effect wasdetected in mixtures containing 701 of antiserum "E" and 30p1 of normal serum (0'~hemolysis). Similar results were obtained with five other batches of antiserum. The volumesof antiserum necessary to inhibit the phospholipase activity of an equal amount of venomvaried from serum to serum depending on its neutralizing potency . In Fig . 1, the minimumvolumes of antiserum necessary to inhibit the phospholipase activity of different amounts ofvenom (up to 16 hr of hydrolysis) were plotted against the amount of venom assayed .Straight lines were obtained whose slopes corresponded approximately to the neutralizingpotencies of the sera, as determined by the pigeon test .

Inhibition of phosphoüpase AZ activity 6y rabbit anti-Crotalus phospholipase serumVariable volumes of rabbit antiphospholipase serum were added to constant amounts of

crude venom and the phospholipase activity of the mixture determined by the egg yolkhemolysistest, in 16 hr hydrolysates. The minimumvolumes ofantiserum necessary to inhibit

Standardization of AnticrotaGc Venom Serum

565

io so 30 +o soVENOAA (Ny)

FIG. I . INHIBr17ON ~ I~iOBPHOLIPASE A= ACI'IVrrY OF C. "WIJ3Y3 ClfrifICU.f VENOM BY HOR.4EANrICROTALIC VENOM SERA WITH DffFFFFREENt' l'O'IENC[F3 ~ NEUTRALIZATION.

Titres given in mg ofneutralizedvenom per ml ofserum, tents carried out in pigeats : A(C-17-77-93)and C(C-28-76A1~ 2.Omg/ml ; D(G28-77-128) and F(G22-77-117 3.5mg/ml ; E(C-23-77-118) andG(C-21-77-113 4 .5 mg/mL Points represent the minimum volume of serum (1 :10 dilution) whichinhibits the enzyme activity oftheceding assayedvenom sample. Hydrolysis time 16hr . Datapoints represent average vahles of duplicate experimentsexcept for sera A andE. Thepoints related tothese two sera represent the mean values of fî-10 experiments. Vertical bars indicate the standard

deviations about the mesas.

566

MARIA HELENA DA SILVA and OTTO GUILHERME BIER

~o so ao so ao'

V E N OIN U~yi

FiG. 2 INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY OF C. dwissus rCffiflCYS VENOM HY RABBITANTIPHOSPHOLIPASE SERUM

Each point represents the minimum volume of antiserum which inhibits the enzyme activity of theassayed venom sample . Hydrolysis time 16hr. Average of two determinations .

TABLE 1 . NEUTRALIZATION OF CRUDE VENOM BY RABBITANTISERUM TO PURIFIED C. durissus terrificus PHOSPHO-

LIPASE A=

Antiserum plus venom mixturesper g mouse

Figures in parentheses are numbers of tnso neutra-lized at that level ofantiserum as estimated by inhibitionof the phospholipase activity ofcorresponding amounts ofvenom

1The LDSO for this batch of C. dratiwus ttrrifrcusvenomwas 0.077 (0.048-0.123) mg,/kg. Teats carried out i .p . inmice.

Antiserum"pl

Venomtpg LDSO

Mortality (24hr)Dead/Total

0.475 (1 .0) 0 .078 1 .0 0/60.475 0.160 2 .1 3/60.475 0.241 3.1 5/60.950 (2 .0) 0 .157 20 0/60.950 0 .241 3.1 3/60.950 0 .321 4 .2 6/61 .425 (3 .0) 0 .257 3.3 0/61 .425 0 .343 4.5 4/61 .425 0 .401 5.2 5/61 .900 (4 .0) 0 .321 4.2 0/61 .900 0 .401 5.2 3/61 .900 0 .471 6.2 4/6

Standardization of Anticrotalic Venom Serum

567

the phospholipase activity were plotted against the venom concentration (Fig. 2~ A straightline was obtained . The "antiphosphofipase potency" was determined by extrapolation to1.0 ml of serum.

Neutralization of venom lethality by rabbit anti-Crotalus phosphotipase AZ serumLethality of mixtures containingvenom plus antiphospholipase serum was tested in mice .

Mixtures were prepared taking into account the volume of serum necessary to inhibit thephospholipase activity ofacertain amount ofcrude venom as determined previously (Fig . 2}Tests were performed at several levels of antiserum andvenomAt a first level of antiserum, avolume of serum sufficient to inhibit the phospholipase activity of an amount of venomcorresponding to 1 Lasso was mixed with venom equivalent to 1, 2 and 3 Lasso . At a secondlevel,a volume ofserum sufficient to inhibitthe phospholipase activity ofvenom equivalent to2 ~sn was mixed with amounts of venom corresponding to 2, 3 and 4 Lasso . Similarprocedure was adopted for twohigher levels ofantiserum . Results are summarized inTable 1 .At the first level of antiserum, the mixture containing 0.475 Etl of serum to 0.078 ~g ofvenomcaused 0~ mortality indicating that 1 Lasso was neutralized . The mixture containing thesame volume of serum to 0.160 hg of venom (about 2 tn so ) resulted in 50'/ mortalityindicating, in agreement with the preceding, that l ~so was neutralized while 1 ip so remainedfree. With 3 i.DSO , venom equivalent to 2 Lasso remained free and caused 100' mortality .Similar results were obtained at the three levels of antiserum.

TABLE 2. NEUTRALIZATIONOF C. 4l1lß3W3 tQITiflgl8 VENOM

BY HORSE AN'rICROTALIC VENOM SERUM A'

Antiserum plus venom mixturesP~ 8 mouse

'Batch C-17-77-93 with a neutralizing potency of2.0mg ofvenom per ml of serum Teats carried out i.v . inpigeon .

tFigures in par~theais are numbers of Lassoneutralized at that level of antiserum as estimated byinhibition of phosphohpase activity of correspondingamounts of venom.$the Loso for this batch of crude venom was 0.077

(0.048-0.123) mg/kg. Tests carried out i.p. in mix .

Antiserum (1 :10)tul

Venom;Wg Las so

Mortality (24 hr)Dead/Total

0.242 (1 .0) 0.078 1 .0 0/60.242 0.157 2.0 3/60.242 0.235 3 .0 6/60.484 (2.0) 0.157 2 .0 0/60.484 0.235 3.0 3/60.484 0.315 4.0 6/60.727 (3.1) 0 .230 3 .0 0/60.727 0.307 4 .0 0/60.727 0.384 5 .0 5/60.969 (4.2) 0 .307 4.0 0/60.969 0.384 5 .0 1/60.969 0 .461 6.0 6/62.358 (10.1) 0.772 10.0 0/64 .716 (202) 1 .543 20.0 2/64 .804 (20.6) 1 .543 20.0 0/6

568

MARIA HELENA DA SILVA and OTTO GUILHERME BIER

TAHLE 3. NEUTRALIZATION OFC. d>Qissu.* ICIIifiCY3 VENOMBY HORSE ANrICROTALIC VENOM SERUM E "

Antiserum plus venom mixturesper g mouse

"Batch C-23-77-118 with a neutralizing potency of4S nlg ofvenom per ml of serum. Tests carried out i .v. inpigeon .

tFigures in parantheses are numbers of I,o soneutralized at that level of antiserum as estimated byinhibition of phophoGpase activity of correspondingamounts of venom.$ The tnso for this batch of crude venom was 0.077

(0.048 - 0.123) mgng. Teats carried out i .p. is mice.

Neutralization of crude venom lethality by horse anticrotalic seraNeutralization tests with anticrotalic sera were performed according to the same

procedure adopted for antiphospholipase serum Results obtained with sera of low and highneutralization potencies are summarized in Tables 2and 3, respectively. Antiphospholipaseactivity and neutralizing capacity of antisera are closely related .

DISCUSSION

Both crotoxin and crude venom exhibit phospholipase AZ activity, indicating that somemolecules of the enzyme are not inhibited by crotapotin. The phophofipase activity isenhanced by normal horse or rabbit serum and is inhibited by specific antiserum againstpurified Crotalus phospholipase A2 or against the whole venom The enhancement of thephospholipase activity by dialyzable constituents of normal serum is attributed to theactivation ofphosphofipase molecules not inhibited by crotapotin. This could also be due toan increased number of free phospholipase molecules resulting from the dissociation ofphospholipase~:rotapotin complexes in the presence of normal serum. Inhibitionexperiments with antisera did not confirm this hypothesis. Thevolumes of antiserum whichcompletely inhibited the enzyme activity were the same, either in the presence or in the

Andserumt(1 :10) ltl

Venom$Rg ~so

Mortality (24 hr)Deadfl'otal

0.133 (1 .0) 0.080 1 .0 0/60.133 0.160 2.1 5/60.133 0.239 3.1 6/60.266 (2.0) 0.160 2.1 0/60.266 0.239 3.1 2/60.266 0.295 3.8 4/60.398 (3.0) 0 .221 2.9 0/60.398 0.295 3.8 3/60.398 0.369 4.8 5/60.512 (3.9) 0.301 3.9 0/60.512 0.401 5.2 3/60.512 0.502 6.5 4/60.683 (5.2) 0 .401 5.2 0/60.683 0.502 6.5 4/60.683 0.602 7.8 4/61 .313 (10.0) 0.772 10.0 0/62.626 (20.0) 1 .544 2R0 0/63.938 (30.0) 2 .316 30.0 2/6

Standardization of Antiaotalic Venom Serum

569

absence ofnormal serumThe dialyzable activating factor maybe related to divalentrationswhich are known to be required for the catalytic activity of the enzyme (BRErrHAVPT,1976) .

Antiphospholipase serum also neutralizes the toxic activity ofcrotoxin (HANASHIRO et a!,1978). Since crotoxin does not dissociate in serum, it is inferred thatantigenicdeterminants ofthe immunogen - the phospholipase molecule - are not significantly affected by itsbonding with crotapotin . Phospholipase complexed with crotapotin can compete withunbound phospholipase for antibody molecules . The neutralization of crotoxin shouldparallel the inhibition of phospholipase. Antiphospholipase serum also neutralizes venomlethality. This was expected since crotoxin is qualitatively and quantitatively the maincomponent of the venom. Experiments with anticrotalic sera have also demonstrated theclose relationship betwcen antiphospholipase activity and neutralizing potency .

These results raise the possibility of titratiog anticrotalic serum in vitro by measuring itscapacity to inhibit the phospholipase activity of the venom Although the method estimatesantibodies to only onevenomcomponent, it is applicable to titration ofcommercial antiserasince it evaluates the neutralization of crotoxin, the component responsible for C. durissusterrificus venom lethality. In addition, hyperimmune sera contain antibodies to all venomcomponents which are neutralized concomitantly with crotoxin . This is inferred since, atleast in the range of venom concentration tested, C. durissus terrificus venom interacts withanticrotalic serum as a system ofonly one component. Further work is required to verify thereliability of the method

REFERENCESBreranr~urr, H. (1976) Enzymatic characteri~tics of Crotahia phospholipase Az and the crotoxin complex. 'Toxicon

14, 221.GoN~ni.ves,J. M. and Vmaw,L. G. (1950) Estudoa sobre venmosde serpentea brasileiraa I . Anélise electroforética.Anu Aced . fires. Cifnc . 22, 141.

Ga~ssetnxN,W. and H~tvw~c, K. (1954) Elektrophoretische Unterwchungen an Schlangen- and Insektentoxinen .Hoppe-Stykr's Z. physiol. Chan. 296, 30.

H~aeat~xr~, E. and Ctu:xGR~unt; D. (1975) Central neurotoxicity ofapamin, aotamin, phoephoGpase Aaad a-amanitin. Toxicon 13, 465.

H~eest~xN, E. and Rüasat~N, K. (1970) Biochemical and pharmacological analysis ofthe socalled crotoxin. InToxinsojAnimaf andPlant Origin, Vol. l, p. 333. Pros of the 2nd International Symposium on Animal and PlantToxins, Tel Aviv . London : Gordon and Breach.

HANASHIRO~ M. A, DA Saves, M. H. and B~ex, O. G. (1978) Neutralization of crotoxin and crude venom by rabbitantiserum to Crotalus phospholipase A. Invnunochemistry 13, 745.

HEnmora, RA. and Fw~Nt~et.-Corrnwr, H. (1971) Biological roles ofthe two oomponenta of~TOtoxin. Proc. natn .Aced. Sci. U.S.A . 68, 1560.

Lrrctn'~, J. T. and wu.coxoni, F. (1949) Asimplified method ofevaluating dose eBcet experiments. J. Pharmac.txp. Thtr. %, 99.

Meal, D. (1970) Acomparative study of enzyme activities in snake venoms . Int. J. Biochtm. 1, 335.Rües~s~t~, K., Hnerrtuurr, H. and H~seautaNx, E. (1971) Biochemistry and pharmacology of the crotoxincomplex. I. Subfiactionation and recombination of the crotoxin complex. Naunyn-Sehmiede6ergs Arch. exp.Path.Pharmak. 270, 274.

Scr~xaeaq S. (1959) Geographical pattern of crotamine distribution in the same rattlesnake subspecies . Science129, 1361 .

Sw~ru, K.H. andFx~tvxn.,-Coxs:~T, H. (1938) Schlangen-gifte. III . Mitteilung : Reinigung undKryatallisation desKlapperschlangen~rittes . Ba. dt. them. Ges. 71, 1076 .