vol.

oo~liolol~~9p3ot-0lo9saa.ao~o

roxteon, 17, x.109-120 . Peryanson PmL Ltd. Printed In Omet Brit .in.

1979.

ANTIBODIES TO SEA ANEMONE NEMATOCYST VENOMII. NEUTRALIZATION OF THE HEMOLYTIC, PHOSPHOLIPASE A~ AND LETHAL ACTIVITIES BY PURIFIED ANTIBODIES PRODUCED IN RESPONSE TO ATTENUATED VENOM D. A . HESSINGER *

and R. I.

GROVEt

"Department of Physiology and Pharmacology, Loura Linda University School of Medicine, Loura Linda, CA 92334, U.S .A . tDepartment of Biology, University of South Florida, Tampa, FL 33620, U.S .A . (Acceptedfor ppbllcatf'on 8 Agqust 1978) D. A. HE$1NGEA and R . I. GROVE. Antibodies to sea anemone nematocyst venom-II . Neutralization of the hemolytic, phospholipase A, and lethal activities by purified antibodies produced in response to attenuated venom. Toxlcon 17, 109-120, 1979 .-The IgG class of antibodies was purified from rabbit serum following the primary immune response to attenuated nematocyst venom from the sea anemone, Alptasla palltda. This immune IgG fraction was judged to be pare immunoglobulin class G by serological and electrophoretic criteria . The amounts of immune IgGwhich specifically inhibited thehemolytic, phospholipase A, and lethal activities of the venom were determined and related to the possible rrxchanisms of hemolytic and mouse lethality. The i.v. mouse ta, n of active anemone venom was 136 kg/kg, making it the most toxic of the reported cnidarian venoms . The implications of the rapid onset of toxic symptoms and the very steep dose-response curve are discussed in terms of the possible mechanism of lethality. INTRODUCTION

ouR previous paper (GROVE and HESSINGER, 1979) we monitored by immunodiffusion techniques the primary and subsequent humoral responses of rabbits immunized with attenuated venom ("toxoid") and active venom prepared from nematocysts (microbasic mastigophores) isolated from the sea anemone Aiptasia pallida. The presence of detectable precipitin reactions between the antisera and the venom preparations suggested a significant level of antibody production . The demonstration that the primary response antisera, elicited toward the attenuated venom, gave identity reactions toward both the attenuated and unattenuated venoms pointed strongly to the attenuated venom being antigenically and immunogenically similar to the native venom. This conclusion was supported by the subsequent response antisera which were elicited by active venom giving identity reactions to both the attenuated and unattenuated venoms . The production of antisera with equivalent serological reactivity toward both the attenuated venom and the unattenuated venom is not only suggestive of a potentially effective antivenin but also of an effective vaccine. Demonstrable neutralization of the venom's known toxicological and biochemical activities is the next Logical step toward assessing the antiserum's effectiveness as a potential antivenin as well as indirectly assessing the toxoids' effectiveness as a vaccine. In order to evaluate the effectiveness of the attenuated venom as a vaccine and to determine its effectiveness in producing antibodies which can neutralize the toxic components of active venom, it was decided to restrict the present study to only the antisera produced IN

109

110

D. A. HESSINGER and R. I. GROVE

toward the attenuated venom (i.e. the primary immune response) . The availability of a nontoxic, yet fully antigenic immunogen is indeed desirable in order to confer successfully active immunity in experimental animals or humans. Since even Lion-immune serum may possess materials capable of nonspecifically affecting some or all of a venom's biological activities (see results), it was decided to use only puri$ed IgG from immune and nonimmune rabbit sera. This approach permits a more accurate assessment of the specific immunological function of the antibodies produced in immunized animals even though only the class G immunoglobulins are studied . In the present paper the neutralization of three of the biological effects of Aiptasia nematocyst venom by immune rabbit IgG is reported . The hemolytic and phospholipase As activities of Aiptasia venom have been described elsewhere (HFSSINGF1t and LENHOTF, 1973, 1974), and the components of the venom to which these activities are attributable have been isolated and partially characterized (HPSSINGER and LENHOFF, 1976) . The toxicity in mice of i.v. injected Aiptasia venom is reported here for the first time. The in vitro neutralization of the phospholipase As, hemolytic and lethal properties of the active venom by immune antibodies (IgG) permits a quantitative assessment of the efficacy of the primary immunization procedure using attenuated venom . The effects of the specific antibodies on these three venom properties are discussed and the amount of antibody which neutralizes these properties is related to the possible mechanisms of hemolysis and lethality . MATERIALS AND METHODS Preparation of xematocyst vetrom Collection and preparation of Aiptasta nematocyst venom was performed as previously described (Hassxotta and Laruroxv, 1973 ; Gtzova and Ht~ssttvcrae, 1979). Venom samples used for the hemolysis, phospholipase and lethality assays in this study were performed from a single venom preparation that was divided and stored frozen at -16°C in a concentration of 435 mg protein/ml . Immunization of rabbits andpreparation of sera Venom attenuation, immunization of rabbits and preparation of immune and pr~immune sera were carried out according to the methods previously described (Gizove and H>_ssnvaex, 1979). DEAE cellulose chromatography of serum Based on results from Ouchterlony immunodiffusion plates, the highest titer antisera from the primary responses of four immunized rabbits were pooled and used for IgG purification . Purification of IgG from immune and non-immune sera was effocted by fiactionation on chromatography columns of diethylaminoethyl~ellulose (DE-52, Whatman, Clifton, N.J .) . Columns were equilibrated at pH 8~0 with 25 mM sodium chloride, S mM Tris-HCl and 001 mM EDTA while the antisera were dialyzed against the same buffer for 24-36 hr, Conductivity and pH of effluent and antisera were monitored to verify equilibration. The absorbency of eluted fractions was monitored at 280 nm . IgG containing fractions eluted from the column without initiating a salt gradient, were identified serologically, pooled, and dialyzed against 50 mM Tris (pH 8~0) and 300 mM glycine. The IgG fraction of pooled pre-immune rabbit sera was likewise obtained . The pooled pro-immune and pooled immune IgG preparations were stored frozen at -16°C until used. Aliquots from the same pooled IgG fractions were used throughout this study. To ascertain the purity of the IgG fractions they were electrophoresed on polyacrylamide gels by the method of Wlu~e et al. (1972) . Prior to electrophoresis the antibody was treated with 40 mM dithiothr+citol and 1 ~ sodium dodecyl sulfate. Piaspholipax array Assays of venom phospholipase activity were performed on a Radiometer pH-stet assembly wnsisting of TTTl titrator and pH meter, SBR-2c Titrigraph rxorder, ABU-lb Auto-burette equipped with a 025 ml burette assembly and a microtitration assembly TTA-31 equipped with a thermostated jacket, VS26 . Temperature of the water jacket was maintained at 30° f 0~1°C with a constant temperature circulating water bath . The titration assembly was equipped with Radiometer calomel electrode type K4112 and glass electrode type G2222C . The standard assay system was essentially Prepared socording to the method of Darus (1973) using Triton X-100 (Rohm and Haas) in a 2:1 molar ratio with lecithin except that egg yolk lecithin (Sigma, type II-E) was used . The reaction was performed at pH 7~7 under nitrogen with a

Neutralization of Nematocyst Venom

111

flasl volume of 2"0 ml and was initiated by the addition of 16 pl of 2"0 M CaCI,. The pH was maintained and the released fatty acids titrated automatically by the addition of 0"01 N KOH. Pirparatlon ojred blood cells axdhernolyNc assay Blood was acquired by cardiac puncture with heparinized syringes from lightly etherized male, SpragueDawley rats . The washed red blood polls were prepared as ~ecribed previously (HPSSINOEA and LBivitoPP, 1973). Hemolytic reactions wen routinely carried out at 30°C as follows: l ml of washed red bloodcells (12"5 vJv) was plead iII a 50 ml Bask with 17 ml of saline-Tris and 2 ml of 110 mM calcium chloride containing 10 mM Trls-HCI, pH 7"4. After addition of venom to the flask, the rate of éemolysis was monitored spxtroP~~e~uY bY withdrawing 1 ml aliquots from the flasks at timed intervals, removing the cells and debris by centrifugation at 4000 g for 30 sec and reading the absorbency (370 nm) of the hemoglobin containing supernatant . Hemolytic activity was measured as the maximum ~ lysis per min. The lytic activity of 4"33 Eig venom per standard 20 ml reaction system was arbitranly defined as one Hemolytic Unit. Eü'ectivoness of the sera and IgG pieparations in neutralizing venom hemolytic activity was studied by incubating diffaont amounts of sera or 1gG with different amounts of venom at room temperature (27°C) for S min in 1 ml plastic tubes. hive minutes of inwbation was suBicient to allow maximal neutralization of venom activity. To initiate hano(ysis the contents of each tube wen transferred to the reaction flasks . The rates of hemolytic wa+e determined as was the amount of antibody needed to neutralize different amounts of venom to one hemolytic unit . Mouse lethality assay Venom lethality was assayed by igjettirig i .v. 0"2-0"3 ml solutions, of different ooIIantrations of venom into the tail vein of groups of female Dublin white mice (25 f 2 g; Flow Laboratories) . The mice were observed for 24 hr and the amount of venom which killed SO % of the mice was defined as the Lo and expi+essed as Eig venom protein pea kg body weight . Groups of four animals were used at one time for each concentration of venom and the assays repeated several times. Neutralization of lethality by antibody (IgG) was assayed by incubating different amounts of IgG with a given amount of venom from S to 12 min before igjection into mice. The amount of IgG which neutralized different amounts of venom to 1 LD was determined. RESULTS

Naturalization of venom hemolytic activity Primary response antisera, produced against attenuated venom, was able to neutralize the hemolytic activity of active venom. Equal amounts of immune and pre-immune sera were incubated for 5 min at room temperature with equal amounts of venom. Although the immune antiserum neutralized 72~ of the hemolytic activity, the pre-immune serum neutralized 42~ of the original activity (Table 1). In an attempt to determine whether the neutralizing action of venom hemolytic activity by pre-immune serum was caused by non-immunoglobulin components of the serum, the IgG fractions of pre-immune and immune sera were isolated by chromatography on DEAE-cellulose columns. TwiuB 1 . NBtTrAAIIZKiSON OP VBNOM ~iBMCILYTIC ACIMTY teiestn~ saAw Arro IgG Vaiomt Venom -I- pre"immune Saum$ Venom -F immune Saum$ Veriomt Venom -F pro-immune Ig(i$ Venom + immune IgG$

HY

PAH-II~NUNE AND

Lyt1C BCtiVity~ 1 "72 1"00 0" SO

Pa+Oent IIeutr811zatlOII 0 42 72

1"83 1"50 0"29

0 18 84

~F.xprr~ed as per neat lysis per min. t8 " 7 Eig protein (LoWAY et al., 1957). $0" 5 mg Protein.

The purity of the DEAF-cellulose IgG fraction was first ascertained on Ouchterlony double diffusion plates by comparing the precipitin reactions of commercially prepared

11 2

D . A . HESSINGER and R . 1 . GROVE

goat anti-rabbit IgG and goat anti-rabbit serum (Miles Laboratories) with both rabbit serum and the purified rabbit IgG fraction (Fig. la). Only one detectable precipitin band appeared between the purified rabbit IgG fraction and the commercial anti-rabbit serum indicating the presence of only one serologically detectable serum component in the purified IgG fraction . The single precipitin band that developed between the purified IgG and the anti-rabbit IgG served to identify serologically the presence of rabbit IgG in the purified fraction. Polyacrylamide electrophoresis in the presence of 1 ~ sodium dodecyl sulfate and 40 mM dithiothreitol yielded only two distinct protein-staining bands corresponding in mobilities to the molecular weights of the heavy and light chains of rabbit IgG (Fig. lb). Immune IgG reduced the hemolytic activity of the venom by 84~, whereas an equal amount of pre-immune IgG exhibited only 18~ neutralization of the original hemolytic activity of the venom (Table 1). The hemolytic activity of Aiptasiu venom varies in direct proportion to and linearly with venom concentrations up to l ug venom per ml. (HLSSINGER and LErHOFF, 1973) The activity in 022 pg venom per ml (i.e. 435 ~g in 20 ml hemolytic assay system) was arbitrarily defined as 1 Hemolytic Unit. To assay the neutralizing activity of rabbit IgG, varying amounts of immune IgG were incubated with 1, 2, 3 and 4 hemolytic units of venom for 5 min before being added to the reaction flasks containing red blood cells. Partial neutralization of the hemolytic activity of Aiptasia venom with immune IgG causes the rate of hemolysis to slow and the prolytic period to increase in duration but does not seem to affect the final extent of hemolysis, which always proceeds to 100 lysis, albeit more slowly. The amounts of immune IgG needed to neutralize the lytic activity by 50~, 60~ and 77% for each of four different amounts of venom were ascertained (such as is shown in Fig. 2 for 1 Hemolytic Unit of venom) . The data for neutralizing all four amounts of venom to 50, 60 and 77~ were consolidated (Fig. 3) as were the amounts of immune IgG needed to neutralize the activity of 2, 3 and 4 hemolytic units to 1 Hemolytic Unit (Fig. 4).

6.0

E

4 .0

T C m U m 3

=

2U

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û

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0.4

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IgG ON VENOM HEMOLYTIC ACTIVITY . Amounts of IgG used in 20 ml are indicated on the abscissa . Amount of venom used was 435 Rg in 20 ml . F10 . Z . EFFECT' OF DIFFERENT AMOUNTS OF llrIMUNE

FIG .

la.

SEROLOGICAL IDENTIFICATION OF FRACi10NATED DIFFUSION .

TgG

BY OUCHTERLONY IMMUNO-

The wells were filled with 200111 as follows : well 1, DE-52 fractionated IgG (2001[g) ; well 2, goat anti-rabbit IgG ; well 3, immune rabbit serum; and well 4, goat anti-rabbit serum. FIG . Ib . SDS POLYACRYLAMIDE GEL ELECIROPHORESLS OF FRACTIONATED IgC. The top of the gel is at the bottom of the test tube . The bands of stained protein correspond to the heavy and light chains of IgG . The amount of fractionated TgG used was 1001[g .

Neutralization of Nematocyst Venom

FIa. 3.

Venom. Ng

COMPARISON OF THH AMOUNTS OF 1gG TO NEUTRALIZE THE HEMOLYTIC ACI7VIIY OF DIFFERENT AMOiJNTS OF VENOM TO SO ~, 6O ~ AND 77 ~.

The amounts of IgG needed to neutralize the hemolytic activity in 433, 8~7, 131 and 174 llg of venom to 50 ~, 60 ~, 9IId 77 ~ were obtained . Data points represent average values of duplicate experiments . All ranges of variation within f 0~1 mg .

Ls

a é

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I/ I/

U' 0

é É

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0.4

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FIO. 4. AMOUNT

1

2

3

,/

4

Hemolytic Units

of IMMiJNE IgG TO NEUTRALIZE THE HEMOLYTIC ACTIVITY OF DIFFERENT AMOtTNTS OF VENOM TO ONE UNIT OF HEMOLYTIC ACTMTY.

Four separate series of experiments were used to determine the amounts of IgG which neutralize 2 and 3 Hemolytic Units to 1 Hemolytic Unit while only one series war used to determine the amount of IgG needed to neutralize 4 Hemolytic Units to 1 . Vertical bars indicate the standard deviations about the means.

Neutralization ofphospholipase activity with immune IgG The ability of immune IgG to neutralize the phospholipase activity of Aiptasia venom was determined by varying the amount of antibody incubated with a certain amount of venom (Fig. S). As with the hemolytic activity (Fig. 2). smaller amounts of immune I¢G are more efficient in neutralizing phospholipase activity than larger amounts (Fig. S). The amount of immune IgG which reduced 50~ of the phospholipase activity in 435 and 6~8 itg ofvenom was 1~93 mg (Pig. ~ and 293 mg (Table 2), respectively.

l16

D. A. I3FSSINGER and R. I. GROVE

ô s

c a 0

0.6

1 .0 Immune IOG, m0

1 .S

Z.0

EFFec,-r OF DIFFER8N4' AMOl7NTS OF IMlSUNE IgG ON VSNO!( PHO!{PHOLIPA38 ACIMrY . The amount of IaG which neutralized S0 ~ of the phospholipase activity of 4ßS ~ of venom is 1"95 mg . Data presented here are value of single experiments . FIO . S.

Neutralisation of venom lethality The mouse 1.D6 p for Alptasfa venom is 3"4 ug venom protein per standardized female mouse of 25 g (Fig. t7 ; that is, 136 pg venom per kg mouse. At venom levels of 2 Ln bp the mice died within minutes and at venom levels of five or more LD6o doses, death occurred within seconds" Mice lethally injected with 2 or less LD6u doses and remaining alive longer than 5-10 sec became lethargic and breathed irregularly . Frequently, partial paralysis of limb, neck and jaw muscles was apparent . Immediately prior to death violent convulsions and spasms of the major skeletal muscles developed with individuals often leaping vertical distances of nearly 1 foot . Slow and labored respiratory movements coupled with gasping and abdominal heaving were the last visible signs before expiration . The intermediate amounts of venom which induce from between 0~ and 100 lethality is narrow ; from 2"6 to 4"3 pg of venom (Fig. 6) . Controls for the buffers used with IgG contained 50 mM Trio and 300 mM glycine at pH 8 and were found to slightly, but significantly, increase the venom toxicity by approximately 0"3 ltg venom per mouse per 0" 1 ml of buffer over the range of 0-0"35 ml buffer . The amount of antibody which neutralized the venom lethal activity of 23 tD 6 p doses (6 "81tg) to 1 I,nap (i .e . 60~ neutralization) was 0"47 mg, and the amount of antibody which reduced 4"3 Ln6o doses of venom (14"5 pg) to 1 r,Dap (i .e. 77% neutralization) was 0"78 mg (Fig. 7) . 1?reimmune IgG in amounts up to 1"5 mg did not detestably neutralize venom lethal activity. DISCUSSION

Antibodies from the primary immune response were selected for use in the present study even though our immunodiffusion data (GROVe and HPSSat~F.R, 1979) indicated that the amounts and diversity of antibodies produced against Aiptasia venom in the subsequent immune responses were greater. Since the antibodies of the primary response were elicited toward the attenuated venom ("toxoid") it was decided that the effectiveness of the toxoid as a potential vaccine could be better evaluated by studying the ability of the primary response antibodies to neutralize some of the de&ted activities of the venom. It was also elected to utilize only the IgC3 fraction of immune rabbit serum rather than

Neutralization of Nematocyst Venom

11 7

u" o~o v"

100

_r

m ô .

nru

~ 60 c m u ro a

al o Z

0

/ °rli ~

Venom. Ng

6

4

FYa. 6. DsrBxe~rsaoN of Douse tn Fox Aipraala vENOM. TheLu  experiment was performed using 25 g female, whiteDublin mice (Flow iaboratorks). Fach venom dose was diluted to 0"2-0"3 ml in physiologic saline and kept at 4 °C. In the fray tlon by each data point tho numerator repres~ts the number of mice mortalities in 24 hr and the denominator the total number of mice injected .

whole immune serum even though the information obtained from such a study would not perfectly represent the total humoral immune response of all immunoglobulin classes toward the toxoid. The purification of serum antibodies for use in this study was necessary to minimis e the problem of nonspecific neutralization ofvenom activities by non-immunoglobulin serum materials (Table 1). Since IgG, however, is the most prevalent class of ~oo

o us

o m `o nrls

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7r17

Immune Igp, mg

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DIFFERENT AAfOLIN1S OF LIMUNE IgG oN ZiIE crl~r rlY of rwo nu~FExENr AlIOUNIa OF VENOM IN AlICE. The two venom wncentrations used were 2"S ~" ° (Curve A) and 49 Ln,o (Curve B) . Venom and IgG were incubated from four to twelve min at 4° prior to i.v . injection into mice . The amount of Igti which neutralize 2"S IA and 4"3 I"O" e to 1 Ln am 0"47 and 0"78 mg, ne9pectively. In the fraction by each data point tho numerator represents the number of mice mortalities in 24 hr and the denominator the total number of mix injected . EFF8C1' OP

serum immunoglobulins and since the Ouchterlony immunodiffusion technique, which detects mostly the interaction of antigen with IgG was used as the means of monitoring the time course of the immune response and estimating the optimum periods of antibody production (GROVE and I3>?ssiNClnt, 1979), it was decided to employ purified rabbit IgG.

118

D. A. HESSINGER and R. 1. GROVE

The i .v. LD go of Aiptasia venom in mice was 136 ltg/kg (Fig. 6) making this venom among the most potent venoms . Lethality of Aiptasia venom is equal to that of the most toxic of the Elapid snake venoms, such as the kraits (Bungarus; FISCHER and KAHARA, 1967), mambas (Dendroaspis ; Sexwlcx and DICKGEISSER, 1963) and the coral snakes ( Micrurus ; RUSSELL and SAULADERS, 1967). Among reported cnidarian nematocyst venoms Aiptasia venom is the most toxic, being between 8 and 50 times more potent than other nematocyst venoms . Stomolophus nematocyst extracts yield an i.v. mouse Lnbo of 5"3 mg protein per kg mouse (Toots and CxnN, 1972). The Lobo for Chrysoara nematocysts has been reported several times, varying from 1 "3 mg/kg (BURNITr and CALTOx, 1973) to 2'0 mg/kg (BuRNETT arid GOLDNER, 1970) t0 2'S mg/kg (BURNETT and GOULD, 1971). Values for Physalia nematocyst extracts have ranged from 0"75 mg/kg (GARRIOTT and LANE, 1969) to 2"0 mgJkg (LANE, 1960). Unfortunately, the several groups working with toxic tentacle and nematocyst extracts from the lethal sea wasp (Chinorex fleckeri) have only reported toxicity bioassays on the mouse in terms of dilutions rather than on the basis ofmg protein so that quantitative comparisons cannot be made. The intermediate range of lethality for Aiptasia venom, between the ma_Yim um nonlethal dose and the minimum lethal dose, is quite narrow, being between 2"6 and 4"3 Ftg of venom per 25 g mouse and corresponding to 0"8 and 1 "3 LD so doses (Fig. 6). BAXTER and MARK (1975) reported that 2 i n6o doses of the sea wasp venom were maximally lethal . Such narrow mortality curves contrast with the broad ranges of mortality for various snake and microbial toxins (IPSEN, 1951 ; CARPENTER, 1975). Broad mortality curves may in some cases be characteristic of toxins which act on very generalized and widely distributed target sites. On the other hand, a narrow lethality range is suggestive of a toxin which acts at very specialized and limited target sites ; sites which probably also express a narrow range of binding affinities . Unlike the lethality of Aiptasia venom to crustaceans (Uca pugilator) which can be primarily ascribed to the low molecular weight, basic protein neurotoxins in Aiptasfa venom (HESSINGER et al., 1973), it has been our unpublished observation that mouse lethality is associated with the more complex hemolytic system of venom proteins (HESsnvcER and LENHOFF, 1976). This is supported by our present finding that the amount of antibody needed to neutralize mouse lethality is quite close to the amount needed to neutralize, to the same extent, the hemolytic activity in the same amount of venom (Table 2). TABLE 2. NELTrRALIZATION OF VENOM ACTIVITY aY

Inhibition 50 60 77

Phospholipase" 2"93 -

Immune IgG, mg Hemolysist 0"17 0"28 a7a

IgG Lethality$ 0"30§ 0"47 o"78

"Amount of IgG to inhibit 50~ of the phospholipase activity (see MATExTAIS and MsrITOr~s) in 6"8 Rg venom. AAmount of IgG to inhibit 50 % of the hemolytic activity in 6"8 flg to inhibit 60 ~ in 8"5 kg, and 77 ~ in 145 Wg venom. The amounts of IgG to inhibit 60 ~ of the hemolytic activity in 83 lIg and to inhibit 77 ~ of the activity in 144 lIg were determined graphically from Fig. 4. $Amount of IgG to inhibit 50 % of the lethal activity in 6"8 fig, to inhibit 60 ~ in 8"5 Elg, and 77 ~ in 14 "5 llg venom. §The amount of IgG to inhibit 50 % of tha lethal activity in 6"8 ug was estimated by linear extrapolation from data in Fig. 7.

Nwtr31i7adon . of Nematocyst Venom

119

Most ofthe lethally envenomated mice die within minutes following injection. The rapidity of Aiptasia venom's lethal effects suggests two things : (1) only one kind of toxin is lethal at the dose range tested ; and (2) the lethal factor is acting on some component of the circulatory system . This latter point is considered reasonable since the time of death following injection would be much longer if the toxic proteins) had to leave the blood to act at more peripheral target sites. For instance, the time of death for mice injected with minimally lethal doses ofbacterial exotoxins is on the order of days (CAxpExrelt, 197 . Hence, either the Aiptasia venom is acting directly on some component of the blood (e.g. to lyse the red blood cells) or the circulatory system (e.g. the heart) . The ratio of mg amounts of antibody to cause 50~ inhibition of lysis to mg amounts of venom protein is about 25 :1 (Fig . 3). At very much higher ratios of antibody to venom the extent of inhibition does not seem to reach 100 but instead asymptotically approaches 90~ inhibition (Fig. 2). This may be explained by either of two mechanisms . (1) It may be that the amity of the antibodies for the hemolytic components ofthe venom is low enough to ensure that there will always be a significant (10~) background of free venom lytic components . Since the antibodies employed in this study were obtained from the primary immune response the affinity of a specific antibody population would be expected to be lower than during the subsequent attamnestic immune responses (Dnvls et al., 1973). (2) On the other hand, since it has also been shown that Aiptasia phospholipase by itself can cause significant levels of hemolytis in the absence of added direct lytic factor (HESSIxc~x and LaNxol~, 1976) and since the level of antibodies specific to the venom phospholipase A$ appears to be quite low in comparison to the amount of antibodies toward the other lytic components of the venom (Table 2), it is likely that the residual low levels of hemolytic activity found in nematocyst venom that has been first incubated with large amounts of specific IgG is due to the action of unneutralized phospholipase AQ . The amounts of immune IgG that neutralize venom phospholipase activity in 6~8 Ng of venom is 17 and 10 times more than the amounts of IgG needed to neutralize the hemolytic and lethal activities of the same amount of venom, respectively (Table 2). At higher amounts of IgG the extent of inhibition of phospholipase activity seems to asymptotically approach 60~ inhibition (Fig. ~. Since the phospholipase Aa activity of Aiptasia venom is totally accounted for by an isozymic pair of acidic proteins (HFSSINGBR and LENx01~, 1976) then the inability of the immune IgG to completely neutralize this enzymic activity of the venom is either due to the elicitation of low affinity antibodies to both isozymes or to the inaccessibility ofthe enzyme's active site to the antibodies. The fairly close parallel between the amounts of antibody to neutralize the hemolytic activity and the lethal activity of Aiptasia venom suggests that the two activities may be causally related to a common venom component or components. Since the level of IgG to neutralize venom phospholipase A$ activity is so much more than the amounts needed to neutralize the hemolytic and lethal activities it can be concluded that the venom phospholipase by itself is not the component responsible for mouse lethality. On the other hand, the synergistic action of the phospholipase with another of the hemolytic components to cause lethality in mice cannot be ruled out by the present data . Preliminary studies with fractionated venom tend to confirm the possibility of more than one hemolytic component, including the phospholipase Ap, being involved in mouselethality(unpublished observation). dcknowled~unmts-This work was supported in part by Immediate Response funds from Florida Sea Grant Program (NOAH) and in part from NIEHS grant ESO 1454-02 .

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D. A. HESSINGER and R. I. GROVE

REFERENCES BAXTEtt, E. H. and Mexx, A. G. M. (1975) Sea wasp toxoid : an immunizing agent against the venom of the box jellyfish, Chironex fleckeri . Toxtcon 13, 423. BtntxcYrr, J. W. and Cat.~rox, G. J. (1973) Purification of sea nettle nematocyst toxins by gel diffusion. Toxicon 11, 243. Buaxsrr, J. W. and Got.n~tt, R. (1970) Partial purification of sea nettle (Chrysaora quinquectrrha) n~atocyst toxin . Proc . Soc. exp. Biol. Med. 133, 987. Baxivsrr, J. W. and Gotnu, W. M. (1971) Further studies on the purification and physiological actions of sea nettle toxin. Proc. Soc. exp. Biol. Med. 138, 759. C~ttrt?ruse, P. L. (1975)In : Immunology andSerology,(3rd Edn.),pp. 251-257. Philadelphia :W. B. Sannders . Dnvts, B. D., DULHEC(,b, R., Etserr, H. N., Gn~aap° a, H. S., WOOD, W. B. and McCex~t~r, M. (1973) In : Microbiology, (2nd Edn.), p. 485. New York : Harper 8c Row. Dears, E. A. (1973) Kinetic dependence of phospholipase A, activity on the detergent Triton X-100. J. Lipid Res. 14, 152. Ftsc~tt, F. G. and K.~,H.~ne, J. J. (1967) In : Animal Toxins, pp. 283, (Russat.t., F. E. and S~uxnxss, P. R ., Eds.). New York : Pergamon Press. G,4xtttoTT, J. C. and LerrE C. E. (1969) Some autonomic effects of Physalia toxin. Toxicon 6, 281. Gaove, R. I. and Ht~stxcex, D. A. (1979) Antibodies to sea anemone nematocyst venom-I. Serological demonstration of specific antibodies produced in rabbits in response to nematocyst venom from the anemone, Aiptasis pallida. Toxicon 17, 99. HPiitt3OER, D. A. and Lt~totrn, H. M. (1973) Assay and properties of the hemolysis activity of pure venom from the nematocysts of the acontia of the sea anemone, Aiptasia pallida. Archs Biochem. Biophys. 159, 629. HtssuvcEx, D. A. and Lt~oFt+, H. M. (1974) Degradation of red cell membrane phospholipida by sea anemone nematocyst venom. Toxicon 12, 379. Hes4txaett, D. A. and Lst~to~, H. M. (1976) Mechanism of hemolysis induced by nematocyst venom: roles of phospholipase A and direct lytic factor. ArchsBiochem. Biophys.173, 603. He~txat?n, D. A., Ll?t~oza+, H. M. and Kettnx, L. B. (1973) Haemolytic phospholipaso A, and nerveaffecting activities of sea anemone nematocyst venom. Nature, New Biol. 241,125. h~v, J. (1951) The effort of environmental temperature on the reaction of mice to tetanus toxin. J. Immwrol. 66, 687. Lwtve, C. E. (1960) The toxin of Physalia nematocysts . Arut . NY.Acad. Sci. 90, 742. LowttY, O., Rost~ttoucx, N. J., F,~xtt, A. L. and Rexneu., R. (1951) Protein measurement with folio phenol reagent. J. Biol. Chem. 193, 265. Rt~ssett, F. E. and S~urmsas, P. R. (1967) In : Animal Toxins. New York : Pergamon Press. Scwttx, G. and Diceostssea, F. (1963) In : Die G(Ji'schlmtgen der Erde, pp . 35-36. Sonderband : Hehringwerke-Mitt . Toots, P. M. and Canx, D. S. (1972) Preliminary studies of nematocysts from the jellyfish Stomolophus mele~rls . Toxicon 10, 605. Wasett, K., Ptetxot.~, J. R. and OSHORN, M. (1972) Measurement of molecular weights by electrophoresis on SDS-Acrylamide gel. In : Methods to Enzymology, Vol. 26, p. 3. New York : Academic Pass.

Antibodies to sea anemone nematocyst venom--II. Neutralization of the hemolytic, phospholipase A2 and lethal activities by purified antibodies produced in response to attenuated venom.

vol. oo~liolol~~9p3ot-0lo9saa.ao~o roxteon, 17, x.109-120 . Peryanson PmL Ltd. Printed In Omet Brit .in. 1979. ANTIBODIES TO SEA ANEMONE NEMATOCYS...
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