Development of specific IgE antibodies after repeated exposure to snake venom

Development of specific IgE antibodies repeated exposure to snake venom Ahmed

A. Wadee, Ph.D., and Arthur

Johannesburg,

after

R. Rabson, M.B., B.Ch., M.R.C. Path.

South Africa

A patient who had been working with snakes for many years developed urticarial lesions on contact with the venom of the poisonous rinkals (Haemachateshaemachatus).More recently, the patient complained of generalized allergic reactions occurring within minutes of exposure to the venom. The patient’s serum, but not control sera, contained IgE antibodies that reacted with the specific snake venom in an ELISA and was demonstrated to associate with a 66 kd component of the venom with Western blotting. With an ELISA, the patient’s serum was also demonstrated to contain IgG antibodies to the specific snake venom and to venom from three other snakes with which the patient had previously been in contact. The possibility of an acute allergic reaction should be considered in individuals continuously working with snakes or in individuals who have previously been bitten by snakes. (J ALLERGY CLIN IMMUNOL 1987;80:695-8.)

Snake venoms are probably the most complex of all poisons and are known to contain a variety of different proteins, each having a multiplicity of effects on numerous organ systems. The best known of these. effects is the neurotoxic activity of many of the venoms of snakes belonging to the family Elapidae or the shock-producing effects of venoms derived from the

family Viperidae.’ Although acute allergic reactions to the antisera used in the treatment of snakebite are well-known, acute anaphylactic reactions causedby direct contact with venom componentsare extremely uncommon. Such an allergic reaction would be expected to occur almost predominantly in individuals continually exposedto snake venom, and this article describesa young man working on a snakefarm who developedacute anaphylactic reactionsafter exposure to the venom of the poisonousrinkals (Haemachafes huemachatus).

METHODS Patient

AND MATERIAL

The patientwas a 25year-old man who had beenworking on a snakefarm for the last 8 years. He had been interested

From the Medical ResearchCouncil Human Cellular Immunology Unit, Department of Immunology School of Pathology of the South African Institute for Medical Research,and University of the Witwatersrand, Johannesburg,South Africa. Received for publication Dec. 4, 1986. Accepted for publication April 2 1, 1987. Reprint requests:A. A. Wadee,Ph.D., S.A.I.M.R., PO Box 1038, Johannesburg2000 South Africa.

SDS-PAGE. Sodium dodecyl sulfate-polyacryl1 Abbreviatioft ‘lde gel electrophoresis 1

in snakes since childhood and had handled rinkals on a number of occasions.His work on the snakefarm entailed handling of many species for demonstration purposes, as well as the collecting of venom from many of the snakes. Although the patient had on two occasionsreceived bites from nonpoisonoussnakes,he had never been bitten by a rinkals. Six years previously, he noticed the development of urticaria on the face and hands when he was handling a spitting rinkals. Lesions occurred only at those sites where venom had been in contact with the skin, and no systemic reactions were described. The patient had been exposedto four episodesof rinkals venom since that time, all during demonstrationsof the spitting ability of the snake. These episodeshad been associatedwith local urticaria at the site of contact with the venom, but more recently, increasing systemic symptomswere described. On the last two occasions of exposure to venom, the patient developedmarked shortnessof breath and acute rhinorrhea and conjunctivitis that developedwithin minutes of exposure.On the last such occasion, thesesymptomswere accompaniedby dizziness, thicknessof the tongue and palate, and acutedyspnea.The patient was advised to changehis occupation. ELISA Venom from the following snakes were supplied by the TransvaalSnakeFarm, Halfway House,Transvaal:Huemachates haemachatus (rinkals), Naja nivea (Cape cobra), Dendraspis polylepis (black mamba),Naja haje (Egyptian 695

J ALLERGY

696 Wadee and Rabson

TABLE 1. IgE antibodies

in the patient’s

serum against

a variety

CLIN IMMUNOL YOVEMaER 1987

of snake venoms

Dihdon

of sacurn

Snake venom

I:10

1:20

1:40

I:50

I.69

Rinkals Black mamba Green mamba Cape cobra Egyptian cobra Spitting cobra

1.012 0.009 0.001 0.001 0.03 0.04

0.447 0.001

0.21 I

0. IO?

0 055 .~ .-

TABLE It. IgG antibodies

in the patient’s

-

serum against

-

various Dhtfon

--

-

snake venoms of serum

Snake venom

1:lO

1:20

1:30

1:40

1:50

Rinkals Black mamba Green mamba Cape cobra

1.677 0.322 0.524 0.182

0.877 0.191 0.415 0.124

0.688 0.166 0.332 0.113

0.467 0.066 0.194 0.058

0.301 0.044 0.123 0.035

cobra), Naja mossambica (spitting cobra), and Dendraspis angusticeps (green mamba). The ELISA was performed in flat-bottomed, 96well microtiter plates (Nunc F Immunoplates I. Roskilde, Denmark). The wells were coated with 100 p,l of various snake venoms at a concentration of 10 kg/ml in sodium carbonate buffer, pH 9.6. Plates were incubated for 1 hour in a humidified chamber at room temperature, after which they were washed four times with phosphate-buffered salineTween, and the unbound sites were blocked with 100 l.~l of 0.5% bovine serum albumin for an additional hour. Plates were washed once more and incubated with 100 ul of varying dilutions of serum from normal volunteers for 2 hours. After an additional wash, the wells were coated with 100 p,l of peroxidase-conjugated goat antihuman IgE or IgG (Miles-Yeda Ltd., Rehovot, Israel) for 1 hour. After a final wash, an additional 100 pl of peroxidase substrate was added to each well, and the reaction was terminated after 15 minutes with 50 pl of 2.5 mol/L of sulphuric acid. Optical densities were read at 420 nmol on a Titertek (Flow Laboratories, MC Lean, Va.) multiscan ELISA reader. In some experiments, the IgE ELISA was performed on the patient’s serum after it had been absorbed with either the specific rinkals venom or with phoapholipase A,, prepared from Naja mocnmbique (Sigma Chemical Co., St. Louis, MO.). In these experiments a 1 in 10 dilution of patient’s serum was incubated in wells precoated either with riukals venom OF with phospholipase A,. After a a-hour incubation period, the absorbed serum was harvested and tested for specific IgE antibodies, as described above.

SDS-PAGE was pe d with 4% stackiug and 10% separating gels as described by Laemmh.’ Qne p& of the gels was stained with Coomassie briRiant blue RZM, and the other part was transferred to a nitrocellulose sheat at 60 V for 3 hours. These latter strips were quenched with 3% gelatin for 1 hour at room temperature and were washed in Tris-buffered saline containing TXveen 20_ There&er~ mmhrane strips were incubated with a l/IO@0 dilution of patient’s or control sera for 2 hours at room tentpcaature with gentle agitation. Membranes were-then washed twice with Tris-buffered salinecontainingTween2@ndiacubstted with a 1: 1000dilution of goat antihuman I.gE hxssmdisiperoxidase conjugate (Sigma Chemical Co.). Co& was developed with a horseradish-peroxidase substrate (B&I&ad; Richmond, Calif.).

Serum from the patient and from various normai laboratory workers was testedon four occasionswith ELBA. On all occasions, signifkant amounts of an IgE antibody againstrink& venom was det%onSrated only in the pat&t’s seem (Table I). I%& tibody could be sp+cifkaMy absorbadwhen pa&~‘s serum was first itIcwith rkrkals venom, and in qhdse experiments, optical density values read,a%420 nm decreasedfrom a mean of I .012 to 0.020 for abskbed serum.

VOLUME NUMBER

80 5

Antibodies

to snake venom

697

pholipase A*, however, had no effect on the level of detectableIgE. IgE antibodiesagainstvenom from six other snakes,including mambasand cobras,could not be demonstrated(Table I), and normal serum did not contain IgE antibodies against any of the snake venoms (results not presented).IgG antibodiesagainstall four venoms were detectedin the patient’s serumand in particularly high titer againstrinkals venom (Table II), but IgG antibodieswere not detectedin numerous control sera. When rinkals venom was separatedon SDS-PAGE, numerous protein bands were observed. When these were transferred to nitrocellulose and incubated with patient’s serum, IgE antibodies were only detected against a 66 kd fraction of the snakevenom (Fig. 1). This protein band was not observed on SDS-PAGE separationsof the five other snake venoms used in this study (results not presented). DISCUSSION Hypersensitivity to snake venom is by no means rare amongpatientswith repeatedenvenomations.Allergic sensitization to rattlesnakevenom was first reported in 1930,3and since that time, venom from all the major snakefamilies have been implicated in the causation of allergic reactions.4 These have chiefly involved the mucus membraneof the eye, nose, and lower respiratory tracts, although systemic anaphylaxis after a rattlesnake bite has been described.5It has therefore been suggestedthat occasional deaths after snakebitesmay be dueto an anaphylacticreaction occurring in an individual who has previously been bitten. The case described in this study is of interest becausethe patient involved had not been bitten by the offending snake, and sensitization, therefore, occurred presumably through repeatedinhalations or repeated contact of the venom with skin or mucous membranes.This resulted in extreme sensitization of the patient, and minute dosesof venom coming into contact with the skin produced whealing at the local site. Ellis and Smith’ previously described a patient in whom systemic anaphylaxis occurred after a rattlesnake bite and in whom skin tests with specific venom elicited a positive response. Schmutz and Stahe16described “anaphylactoid” reactions after snakebitesand suggestedthat these could contribute to fatal reactions occurring in individuals who had previously been bitten. The patient described in this article had true anaphylactic reactions, and IgE antibodies to specific rinkals venom could be identified with an ELBA. The antibody could be absorbedout by incubating it

FIG. 1. SDS-PAGE of rinkals venom (right tection of IgE antibody to the 66 kd fraction blot analysis (left lane).

lane) and dewith Western

with pure rinkals venom but not with venoms from a variety of other snakes (results not presented) or phospholipase A2 derived from Naja mocambique. The rinkals venom could be separated out with SDS-PAGEinto numerous proteins, ranging in molecular weight from 21 to 200 kd. The IgE antibody in the patient’s serumwas demonstratedwith Western blotting to react with a 66 kd fraction of the rinkals venom. This fraction was not present in a variety of other snakevenoms, explaining the lack of IgE reactivity againstthese other venoms. Of interest was the finding of IgG antibodiesin the patient’s serumagainst three other snakevenoms to which the patient was in

Wadee and Rabson constant contact. Similar IgG antibodies could not be detected in the serum of numerous normal individuals. The ability to raise IgG antibodies to snake-venom components does raise the possibility of treating the allergic patient with hyposensitization therapy in a manner analogous to the treatment of patients allergic to bee stings. However, the extreme toxicity of this venom probably makes this approach unreasonable. The documentation of this allergic reaction may be of importance to the patient because an immediate collapse reaction after a future snakebite may be of allergic causes, and treatment of this allergic emergency could be lifesaving.

J ALLERGY

CLIN. IMMUMOL NOVEMBER 1997

REFNlENcES

I. Minton SA. Snake venoms and envenomatlon. New York: Matccl Dekker Inc. 1971:43-86. 2 Laemmli UK. Cleavage of structural pro&ems dunng assembly of the head of bactertophage T4. Nature 19701127~?80 3. Zozaya J, Stadelman RE. Hypersensttiveness to bnake venom proteins. case report. BuIl Antivenin Inst America 193fk3.91 4. Parrish HM. Pollard CB. Effect of repeated polsonou.\ snake bites In man. Am J Med Sci 1959:237:?77. 5 Ellis EF, Smtth RT. Systemic anaphylaxls dfter rattle snake trite JAMA 1965; 193&l 6. Schmutz J, Stahel E. Anaphylactold reacttons~to Lnake bltr Lancet 1985:2 1306.

Anaphylactoid reactions (ARs) occurring in patients receiving propranolol have been described as unusually severe and having a “sluggish” response to epinephrine. Although the mecham’sm of MS to iodinated radiographic conrrast media is not IgE mediated, because of wtiqxead use of P-adrenergic blocking agents, we undertook a prospective study to determine the incidence of AR to radiographic contrast media during cardiac angiography. Nine hundred j@y-rwo consecutive patients were divided into four groups according Foconcomitant chronic medicaiioas. Group I (447 patients) were receiving no &zdrenergic blocking agents or calcium antagonists. Group II (216 patients) were receiving a /3-adrenergic blocking agent. Group 111(147 patients) were receiving a calcium antagonist but not a Padrenergic blocking agemt. Group IV (I42 patients) were receiving both a calcium antagonist and a P-adrenergic blocking agent. The reaction rates, respectively, in the four groups were 4.476, 7.41%, 5.44%, and 4.93%. The rates of ARs were not associated with the use bf concomitanF medications in any of the groups (2 = 2.531; p = 0.47). The probabilio of a c>pe II error in comparison of groups I and II was 0.75 should the true incidence of reactions in patients receiving @adrenergie antagonists be 7.41%. No d@erence in the incidence of AR was observed between patients taking seiective and nonselective p-adrenergic blocking agents in group II. Spec@ic ARs occurring in parients receiving P-admnergic blockiug agents were usua& t&d and often without need for specific pharmacotherapy. (J ALuRGY CUN IWIU~~O~1987;8&698-702.)

From the Sections qf Allergy-Immunology and Cardiology, Department of Medicine, Northwestern University Medical School, Chicago, Ill. Suppo&ed by United States Public Health Service Grant AI 11403 and the Ernest S. Bazley grant. Received for publication Sept. 21. 1986.

epted for p&liiion May 5, 1!?87. I?kprint requests: Paul A. Greenberger, M.D.. 383 E Chqgo Ave., Chicago, IL 60611. *Statistical advice was provided by Carl B. Wal&ark, MS., I%hmtry !&&on, Cance&enter, No&~kres+ern %&&y&&dical School, Chicago, 111.