CLINICAL TOXICOLOGY, 29(4), 493-503 (1991)

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DIAGNOSIS OF SNAKE VENOMS BY A REVERSE LATEX AGGLUTINATION TESTS

Leera Chinonavanig, Ph.D.*, Chul Karnchanachetanee, M.D.**, Pattaya Pongsettakul, M.D.***, Kavi Ratanabanangkoon, Ph.D.* Department of Microbiology, Faculty of Science, Mahidol University, Bangkok*; Chaopraya Aphaiphubet Hospital, Prajinburi Province**; Sadao Hospital, Songkla Province***, Thailand

ABSTRACT A reverse latex agglutination test using protein A column purified rabbit

antivenom IgG-sensitized latex particles was developed for the detection of the six medically important snake venoms of Thailand. The detection limit of the reverse latex agglutination test was 0.16 to 1.2 pg/mL of crude venoms. Cross-reactions with heterologous venoms were observed at concentrations 460 to 16000 times that of homologous venoms. Detection of various snake venoms in clinical specimens was carried out by the reverse latex agglutination test. The sensitivity was 52.5% of the 59 serum samples. There was one (1.69%) false positive sample. The positive detection of venom in wound swabs (26 cases) was 38.5% and was not statistically different from that observed in paired serum samples. Interference from human plasma, serum and urine on the reverse latex agglutination test could be eliminated by adsorption with normal rabbit IgG-coated latex suspension

$Address reprint requests to: Dr. Kavi Ratanabanangkoon, Department of Microbiology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. 493 Copyright

@

1991 by Marcel Dekker, Inc.

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or by heat inactivation at 56OC for 30 min. Prozone effect observed in some sera was eliminated by heat inactivation at 56OC for 30 min. The sensitized latex particles were stable at 4OC and -2OOC for at least 3 months. Cycles of freezing-thawing and lyophilization did not change their reactivities. The total test time was about 40 min. (Key Words: immunodiagnosis; snake venom; reverse latex agglutination.) INTRODUCTION Snake venom poisoning remains an important medical and economic problem in Thailand and many other developing countries, mostly in tropical regions. The way of life, together with the abundance of several species of poisonous snakes, make the people of these countries highly vulnerable to snakebite. The major problem in treatment, apart from the usual delay in reaching a rural health center, is the need for accurate diagnosis of the source of envenomation (1,2). Various snake venoms produce similar local and systemic effects that are not useful for diagnostic purposes (3). Species diagnosis is essential in countries such as Thailand where only monovalent non-cross neutralizing antivenoms (4) are available. ELISA is probably the most popular immunoassay developed for the purpose of snake venom diagnosis with some advantages in sensitivity (5,6). However, some versions of ELISA in current use still lack required specificity (7,8), are too slow for treatment (3) or are too expensive for use in developing countries (9). In addition, the reagents are often not sufficiently stable for storage and transportation conditions in tropical areas. The present study involved the development of a simple and inexpensive test based on reverse latex agglutination (RLA) for rapid species diagnosis of snake venoms of Thailand.

MATERIALS AND METHODS Venoms and Chemicals Six major poisonous snake species for which horse antivenons are available in Thailand were studied. They were Naia naia siamensis or m a

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kaouthia, Bunearus fasciatus and Ophiophapus hannah of the ElaDidae family, ViDera russelli, Calloselasma rhodostoma and Trimeresums albolabris of the ViDeridae family. The snakes were kept and milked at the Queen Saovabha Memorial Institute, Bangkok. Each venom was collected from 3-8 snakes of the same species, pooled and immediately frozen on dry ice. Extreme care was taken to avoid cross-contamination between these venoms. The venoms were stored at -20°C until used. Polystyrene latex particles (0.822 micron) were obtained from Dow Chemical Co. Chemicals were of reagent grade and were purchased from Sigma Chemical Co., St. Louis, MO, unless otherwise indicated. Rabbit Antisera Antiserum to each of the six snake venoms was produced by immunization of rabbits with venom as described by Chinonavanig et al. (10). The rabbit antisera were kept at -2OOC until used. Purification of Rabbit Immunoerlobulins Rabbit immunoglobulin was precipitated from whole antisera by neutral salt fractionation using saturated ammonium sulfate solution (1 1). The immunoglobulin was further purified by Sepha-rose 4B-Protein A affinity chromatography using ice cold 3 M sodium thiocyanate as an eluting agent (12). The eluted immunoglobulin G (IgG) was used in the sensitization of latex particles. Reverse Latex Arrglutination (RLA) Test 1. Coating of purified antivenom IgG to latex particles Latex particles were sensitized with purified IgG against each of the six venoms as described by Hudson and Hay (13) with some modifications. A 10%suspension of polystyrene latex particles was washed twice in 0.1 M glycine buffer saline pH 8.2 (GBS), by centrifugation at 9650 x g for 5 min and then prepared as a 0.8% latex suspension in 0.1 M GBS, pH 8.2. The coating was performed as follows: three mL of an optimal concentration of IgG were mixed with 120 p1 of a 0.8%latex suspension. The mixture was incubated in a 37OC water bath with shaking for 30 min, then washed twice in 0.1 M GBS, pH 8.2. The sensitized latex particles were resuspended in

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240 pl of diluent (0.1 M GBS, pH 8.2 containing 0.1% bovine serum albumin) to give a 0.4% latex suspension stored at 4OC until used. 2. Detection of snake venoms by the RLA test The RLA test was performed on an eight circle, dark background glass slide. Each of the six snake venoms was serially diluted in the diluent 0.025 mL of the venom was then mixed with 0.025 mL of the antivenom IgG-sensitized latex suspension. The slide was rotated gently with a mechanical rotator for 5-10 min. The result was read with the naked eye under a high-intensity light source. A test was positive when visible agglutination was observed. The venom titer was expressed as the reciprocal of the highest dilution giving complete agglutination. The diluent control consisted of 0.025 mL of the diluent added to 0.025 mL of the immune IgG-sensitized latex suspension. The sample control was composed of 0.025 ml of venom mixed with 0.025 mL of normal rabbit IgG-sensitized latex suspension. These negative controls had to show no agglutination.

3. Detection of snake venoms in clinical specimens by the RLA test

Two hundred mL of each clinical sample were adsorbed with an equal volume of a 5 % suspension of normal rabbit IgG-coated latex particles. The mixture was rotated at room temperature for 30 min. After incubation, the latex was removed by centrifugation at 9650 x g for 5 min. To 0.025 mL of serial two-fold dilutions of the adsorbed sample, beginning with a 1:2 dilution, 0.025 mL of one of the six antivenom IgG-sensitized latex suspensions were added. The reactions were mixed gently for 10 min before the results were read. Clinical SPecimens Clinical specimens were collected from all patients with snakebite regardless of the severity of the bite or the occurrence of systemic poisoning. All of the patients brought the culprit snakes for identification. Twenty-eight sera were obtained from four cases of N. n. siamensis bites, 19 cases of V. russelli bites and five cases of T. albolabris bites. In addition, 31 serum and

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26 wound swab samples were gathered from patients bitten by C. rhodostoma, including 24 paired serum and swab samples from the same patients. Seventeen serum samples were kind gifts from Dr. Sasithon Pukrittayakamee. The sera and wound swabs were stored frozen at -2OOC until tested. The wound swab samples were soaked with 1 mL of 0.85% normal saline solution and the cotton swabs were removed prior to assay. Control human plasma, serum or urine was studied for interference with the RLA test. Thirty three plasma, 40 sera and 31 urine samples were obtained from rural patients who came to hospitals without a history of snakebite. The plasma and serum samples were stored at -2OOC until used. The urine samples were stored at 4OC and examined within seven days of collection. All samples were centrifuged at 500 x g for 10 min to remove insoluble material before the test. Statistical Analysis The differences between sample sources used in the detection of snake venoms by the RLA technique was analyzed by means of the Chi square test Xz,McNemar Test).

RESULTS Detection of six snake venoms by the RLA test Table 1 indicates the relative sensitivity and specificity of venom detection by the RLA test. The antivenom IgG-sensitized latex particles were agglutinated in the presence of the corresponding homologous venoms at concentrations ranging from 0.159 to 1.221 pg/mL, depending on the snake venom. Cross-reactions were observed with heterologous venoms at concentrations ranging from 162 to 3700 pg/mL or 460 to 16OOO times higher than those of the homologous venoms.

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CHINONAVANIG ET AL. TABLE 1 Sensitivity and Cross Reaction of the RLA Test of the Six Snake Venoms

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Venom N.n.siamensis (NS) B.fasciatuS @F) 0.hannah (OH) C .rhodostoma (CR) V.russelli (VR) T.albolabris (TA)

Detection limit (Ag) dmL 0.879 0.226 0.317 1.221 0.159 0.352

Cross-reacting venom &g/mL) NS

BF

225 2200 2200 >2200 2200

1850

OH

CR

~ 2 6 0 0 >1300 1300 >1300 925 650 3700 >2600 925 2600 >1300 1850 >2600 1300

VR

TA

>1300 >2000 162 2000 >1300 >2000 >1300 2000 760 162

Interference in clinical samDles Autoagglutination was occasionally observed when the control human plasma, serum and urine samples were assayed by the RLA test. This interference could be eliminated by adsorption of the samples with normal rabbit IgG-sensitized latex suspension at room temperature for 30 min or by inactivation of the sera at 56OC for 30 min. However, addition of normal rabbit serum or IgG to clinical samples prior to or during agglutination reaction did not completely eliminate the interference. Stabilitv of the antivenom IeG-sensitized latex sumension The antivenom IgG-sensitized latex particles stored at 4OC as a 0.4% suspension in 0.1 M GBS, pH 8.2 containing 0.1% bovine serum albumin were stable for at least 2-3 months. After this period, the titer of agglutination for the corresponding antivenom IgG-sensitized latex particles was reduced. The immune IgG-sensitized latex suspension was stable for 3 months at -2OOC. Nonspecific agglutinations occurred after longer storage. The immune IgG-coated latex particles were stable to at least 15 cycles of freezing-thawing and to lyophilization, with no change in their agglutination properties.

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Detection of venoms for species diagnosis in clinical specimens bv the RLA test Of the 59 serum samples, 31 were identified by the RLA test: 3/4 cases of N,n. siamensis, 17/31cases of C. rhodostoma, 10119 cases of russelli and 1/5cases of T. albolabrig. Ten of the 26 swab samples were positive for C . rhodostoma. The snake venom titers of the clinical samples reactive with the RLA are shown in Figure 1. Five cases of C. rhodostoma bites gave partial cross-reactions with N.n. siamensis and B. fasciatus antivenom IgG-coated latex particles. One sample assumed to be T. albolabris snowed a false positive reaction for V. russelli venom. All sera of 27 patients bitten by C . rhodostoma from Sadao Hospital gave a prozone-like effect unless they were diluted 8-16fold. However, the prozone effect disappeared after inactivation of the serum samples at 56OC for 30 min. ComDarison of serum and wound swab samules used for the detection of C. rhodostoma venom bv the RLA test Table 2 compares the results for venom detection in sera and wound swabs by the RLA test. Of the 24 paired serum and swab samples, seven cases were dual positives. There were seven cases of positive detection only in the sera and three cases of positive detection only in the swab samples but the detection of venom from the two sample sources was not statistically different (p > 0.05).

DISCUSSION Various immunodiagnostic tests have been developed for the detection of snake venoms (1,7). Although the sensitivity of the present RLA test was at 0.159 to 1.221 pg/mL venom, the positive detection of various snake venoms in clinical specimens obtained, regardless of the severity of the bite, was 52.5% . This figure compares favorably with the ELISA results of 39 % for acute bites by N.n. siamensis and B. fasciatus (3), but somewhat less favorably with ELISA results of 97% for systemic poisoning cases by rhodostoma (14). The specificity of the RLA test was reasonably high. Only

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a 0.

0

.

AAAAA

. *o: :

1

0.0. 00.0

I siamensis rhodostoma russelli albolabris ( 17/ 31.)

Figure 1.

Venom titers by the RLA test using as source of antigen either serum ( 0 ) or wound swab (A) samples from patients bitten by various snakes of Thailand.

TABLE 2 Comparison of 24 Paired Serum and Swab Samples from the Same Patients in the Detection of C. rhodostoma Venom by the RLA Test Serum Positive Negative Total

Positive 7

3 10

p-value > 0.05 by X2 McNemar Test

Swab Negative 7 -7 14

Total 14 10 24

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one (1.69 96) of 59 serum samples gave a false positive reaction. Five cases of positive C . rhodostoma venom detection showed weak partial crossreactions with N.n. siamensis and B, fasciatus antivenom IgG-sensitized latex particles. These inter-family cross-reactions pose less of a problem in the identification of snake species since the clinical signs and symptoms produced by C.rhodostoma venom differ from those caused by N.n. siamensis and B, fasciatus venoms. In the RLA test, non-specific agglutinations by normal human sera were observed, mostly with sera of rural people (D.A. Warrell, personal communication). The prozone-like effect observed in serum samples obtained from Sadao Hospital was attributed to the collection and preparation of the sera. However, heat inactivation of the sera and wound swab samples eliminated the non-specific agglutinations and the prozone effect. In addition, the weak reactions shown by some samples in the RLA test became strongly positive after heat treatment. Although the sensitivity of the RLA test in the paired wound swab samples (10/24, 41.7%) was lower than that in the paired sera (14/24, 58.3%) the difference was not statistically significant (p > 0.05). Unfortunately dry swabs, which are known to contain less venom than wet ones (15), were collected for this study. It is speculated that if wet swab samples had been collected and examined by the RLA test, a greater number of positive venom detections would have been registered. The main advantage of using wound swabs for the detection of snake venom is that they can be easily and rapidly obtained from bite sites. The stability and relative simplicity of the RLA test are appropriate to the storage, transportation and laboratory facilities of tropical and developing countries. The RLA method is inexpensive (US $0.05 per test for RLA cf. US $0.50 for ELISA at Mahidol University, excluding the cost of antigen and antibody which can vary widely). It requires a one-step antigen-antibody reaction and is completed in 40 min. The RLA test, with some improvements in the sensitivity, should be useful for snake venom diagnosis in rural hospitals.

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ACKNOWLEDGEMENT

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The study was supported by grants from the National Research Council of Thailand and the United States Agency for International Development (USAID; Grant No. 936-5542-G-00-5079-00). The authors thank Drs. Sasithon Pukrittayakamee, Pornchai Matangkasombut and T.W. Flegel for valuable suggestions and assistance.

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Theakston RD. The application of immunoassay techniques, including enzyme-linked immunosorbent assay (ELISA), to snake venom research. Toxicon 1983;21:341-352. Ratanabanangkoon K, Billings PB, Matangkasombut P. Immunodiagnosis of snake venom poisoning. Asian [email protected] J Allergy Immunol 1987;s: 187-190. Viravan C, Veeravat V, Warrell MJ, Theakston RD, Warrell DA. ELISA confirmation of acute and past envenoming by the monocellate Thai cobra JNaia kaouthia). Am Trop Med Hyg 1986; 35:173-181. Ganthavorn S. Toxicities of Thailand snake venoms and neutralization capacity of antivenin. Toxicon 1969;7:239-241. Theakston RD, Lloyd-Jones MJ, Reid HA. Micro-ELISA for detecting and assaying snake venom and venom antibody. Lancet 1977;2:639-641. Coulter AR, Harris RD, Sutherland SK. Enzyme immunoassay for the rapid clinical identification of snake venom. Med J Aust 1980;l: 433-435. Minton SA. Present tests for detection of snake venom: Clinical application. Ann Emerg Med 1987;16:932-937. Ho M, Warrell MJ, Warrell DA, Bidwell D, Voller A. A critical reappraisal of the use of enzyme-linked immunosorbent assays in the study of snake bite. Toxicon 1986;24:211-221. Chandler HM, Hurrell JR. A new enzyme immunoassay system suitable for field use and its application in a snake venom detection kit. Clin Chim Acta 1982;121:225-230.

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Chinonavanig L, Billings PB, Matangkasombut P, Ratanabanangkoon K. Antigenic relationships and relative immunogenicities of venom proteins from six poisonous snakes of Thailand. Toxicon 1988;26: 883-890. Heide K, Schwick HG. Salt fractionation of immunoglobulins. In: Handbook of Experimental Immunology Immunochemistry. Weir DM, ed., 2nd ed, Vol 1, Oxford: Blackwell Scientific Publications, 1973: p 6.1. Goding JW. Use of staphylococcal protein A as an immunological reagent. J Immunol Methods 1978;20:241-253. Hudson L, Hay FC. Practical Immunology, 2nd ed, Oxford: Blackwell Scientific Publications, 1980:137. Ho M, Warrell DA, Looareesuwan S, Phillips RE, Chanthavanich P, Karbwang J, et al. ClinicaI significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma). Am J Trop Med Hyg 1986;35:579-587. Morrison JJ, Pearn JH, Coulter AR, Halliday WJ. Immunological stability of an elapid venom, Tropidechis carinatus and its relevance to the clinical detection of snake venom. Am? J Ejcp Biol Med Sci 1983;61:489-495.

Diagnosis of snake venoms by a reverse latex agglutination test.

A reverse latex agglutination test using protein A column purified rabbit antivenom IgG-sensitized latex particles was developed for the detection of ...
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