Biologicals 42 (2014) 8e21

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Neutralization of cobra venom by cocktail antiserum against venom proteins of cobra (Naja naja naja) C. Venkatesan a, M. Sarathi a, b, G. Balasubramanaiyan a, c, S. Vimal a, N. Madan a, N. Sundar Raj a, S. Mohammed Yusuf Bilal d, A. Nazeer Basha a, M.A. Farook a, A.S. Sahul Hameed a, *, G. Sridevi e a

Aquaculture Biotechnology Division, Department of Zoology, C.Abdul Hakeem College, Melvisharam 632 509, Vellore Dist., Tamil Nadu, India Department of Biology, Lakehead University, Thunder Bay, ON P7B 5E1, Canada Department of Zoology, Arignar Anna Govt Arts College, Cheiyar, Tamil Nadu, India d Raja Muthaih Medical College, Annamalai University, Chidambaram, India e King Institute of Preventive Medicine, Chennai, Tamil Nadu, India b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 27 May 2013 Received in revised form 6 September 2013 Accepted 7 September 2013

Naja naja venom was characterized by its immunochemical properties and electrophoretic pattern which revealed eight protein bands (14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa) by SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The results showed that Naja venom presented high lethal activity. Whole venom antiserum or individual venom protein antiserum (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) of venom could recognize N. naja venom by Western blotting and ELISA, and N. naja venom presented antibody titer when assayed by ELISA. The neutralization tests showed that the polyvalent antiserum neutralized lethal activities by both in vivo and in vitro studies using mice and Vero cells. The antiserum could neutralize the lethal activities in in-vivo and antivenom administered after injection of cobra venom through intraperitoneal route in mice. The cocktail antiserum also could neutralize the cytotoxic activities in Vero cell line by MTT and Neutral red assays. The results of the present study suggest that cocktail antiserum neutralizes the lethal activities in both in vitro and in vivo models using the antiserum against cobra venom and its individual venom proteins serum produced in rabbits. Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Keywords: ELISA MTT Neutral red Naja naja Cocktail antiserum Cobra

1. Introduction Snakebite is a serious problem in tropical and subtropical countries and responsible for morbidity and mortality. Global incidence is estimated to be about 5.4 million snakebites per year [1]. Snakebite is also a major public health problem and claims a large number of lives in the Indian subcontinent. In India, approximately 15,000e20,000 people are affected every year by snake envenomation [2]. The four major poisonous such as snakes cobra, krait, Russell’s viper and saw-scaled viper are responsible for fatality in India. The venoms of cobra and krait of Elapidae family are neurotoxic in nature, considered to attack the victim’s central nervous system and usually results in heart failure. Immunotherapy using polyvalent antivenom is the only effective treatment against snake venom poisoning. Antivenoms are usually prepared by immunizing large animals, usually horses, with individual venom * Corresponding author. Tel./fax: þ91 4172 269487. E-mail address: [email protected] (A.S. Sahul Hameed).

or a range of different venoms obtained from several snakes to eliminate intraspecific variation [3]. Anti-snake venom therapy may cause various side effects such as anaphylactic shock, pyrogen reaction and serum sickness [4]. These side effects may be due to the presence of high concentrations of non-immunoglobulin proteins in many commercially available antivenoms [5]. Moreover, antivenom production in animals is time consuming, expensive and requires ideal storage conditions. In view of the above facts, an alternate technology for production of antivenom has to be developed or a suitable snake venom neutralizing agent has to be found [6]. In the present study, an attempt was made to develop an alternate technology to neutralize the venom of Indian cobra using cocktail antiserum prepared by mixing of antisera raised against individual proteins of venom of cobra and we report the production of species specific antiserum, neutralization of its lethal actions by species specific antiserum raised against Naja naja venom using rabbit. The results of this study will form the basis for development and application of monoclonal antibodies to treat envenomation.

1045-1056/$36.00 Ó 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2013.09.002

C. Venkatesan et al. / Biologicals 42 (2014) 8e21

2. Materials and methods

9

was dialyzed and concentrated by a Speed vac evaporator. The purified venom proteins were estimated and confirmed on SDS-PAGE.

2.1. Collection of snake venom 2.6. Detoxification and production of antiserum The lyophilized snake venom was collected from cobra, and obtained in Irula Snake Catcher’s Society, Chennai, Tamil Nadu, India with proper permission (No.WL1/7/2009, dt 13.11.2008) and the sample preserved in desiccator at 4  C for further use. It was dissolved in 0.9% saline and centrifuged at 2000 rpm for 10 min. The supernatant was collected and kept at 4  C until further use. Venom concentration was expressed in terms of dry weight and protein concentration determined by Lowry method [7]. Commercial polyvalent antiserum was purchased from King Institute of Preventive Medicine, Chennai, Tamil Nadu, India. 2.2. Collection and maintenance of experimental animals New Zealand rabbit (2e3 kg), adult Swiss mice (20e22 g) and Balb/c mice (18e22 g) obtained from the Institute of Veterinary Preventive Medicine (IVPM), Ranipet, Tamil Nadu, India were used in this study. They were kept in animal cages with sawdust as bedding under conditions of 12:12 h light and dark cycle and fed with standard diet. Equal numbers of male and female mice were used in each experimental group, keeping their mean weight as near as possible. The animal studies were conducted with the prior permission of the Institutional Animal Ethics Committee, C.Abdul Hakeem College, Melvisharam, Tamil Nadu, India (No.1011/c/06/ CPCSEA, dt 19.12.2006). 2.3. Collection and maintenance of Vero cell line The Vero cell line (Ethiopian kidney green monkey cells from American type culture collection CCL81) was obtained from King Institute of Preventive Medicine, Chennai and was used to study the neutralization of cytotoxic effects of N. naja venom. The cells were cultured in 25 cm2 culture flasks in DMEM, with 10% fetal bovine serum (FBS) and 2% of antibiotic solution (100 mg/ml streptomycin and 100 IU/ml penicillin) incubated in a CO2 incubator with 5% of CO2 at 37  C. 2.4. SDS-PAGE analysis of snake venom Snake venom protein is medicinally important and analyzed by 12% SDS-PAGE under reducing conditions [8]. Prior to electrophoresis, venom samples (10 mg) were mixed 1:3 (v/v) with Laemmli sample buffer (10% SDS, 10% w/v b-mercaptoethanol, 50% sucrose, 0.02% bromophenol blue), boiled for 5 min, and electrophoresed at a constant current of 30 mA. After electrophoresis the gels were stained with Coomassie Brilliant Blue. Molecular weight standards were co-electrophoresed. Phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soyabean trypsin inhibitor (92 kDa) and lysozyme (14.3 kDa) were used as colored molecular weight markers. 2.5. Electroelution of different proteins of N. naja venom The major venom proteins were electro-eluted on the basic method [9]. A preparative SDS-PAGE was run with proteins of cobra venom. After the run, the gel was soaked in prechilled KCl (0.4 M). The prominent venom protein bands were excised and the gel slices were minced into small pieces (w1 mm) using a sterile razor blade. The gel pieces were transferred into a dialysis bag with TE buffer (10 mM TriseHCl and 1 mM EDTA, pH 8.0) and the bag was kept in a horizontal electrophoretic tank filled with TE buffer. Constant power supply (50 mA) was set and run for 6 h. After elution the sample

Indian cobra venom (1 mg/ml) was dissolved in physiological saline. The dissolved venom was detoxified by heating at 60  C for 60 min and was immediately followed by placing the sample in icecold (0e2  C) water for 10 min [10]. This treatment was repeated twice. Male healthy New Zealand white rabbits (2e3 kg body mass) were chosen for the production of polyclonal antibodies. Detoxified cobra venom and individual venom proteins (200 mg/kg of body mass) were emulsified with an equal volume of Freund’s complete adjuvant and injected intramuscularly at multiple sites. First booster dose (200 mg/kg of body mass) was given along with Freund’s incomplete adjuvant intramuscularly, after 4 weeks of first dose [11]. After first booster dose, injections i.e. second and third booster doses (200 mg/kg of body mass) were administered intramuscularly along with Freund’s incomplete adjuvant at an interval of 2 weeks [12]. The blood was collected from the marginal ear vein. After coagulation, the blood was centrifuged at 2000 g for 10 min and the serum was collected for determination by ELISA and Western blot analysis [13,14]. 2.7. Western blot analysis Cobra venom (20 mg) was first fractionated by SDS-PAGE, as described above. The gel was placed in the electroblotting apparatus adjacent to nitrocellulose paper in buffer, as described by Towbin and his co-workers [15]. After transfer, the nitrocellulose paper (NCP) was blocked for 1 h with 3% skimmed milk in PBS (20 nM sodium phosphate containing 0.9% NaCl, pH 7.2). The NCP was washed in PBS for 5 min and then incubated with 1:10,000 dilution of rabbit anticobra antiserum (or) rabbit antiindividual venom proteins antiserum for 1 h. This membrane was washed three times in PBS containing 0.05% Tween-20 (PBS/T) followed by PBS was three times for 20 min each. The membrane was incubated with 1:30,000 dilutions of alkaline phosphatase conjugated goat anti-rabbit IgG for 2 h. The membrane was washed as described above and developed with the substrate nitroblue tetrazolium and 5-bromo-4-chloro indolyly phosphate in substrate buffer 10% 1 M Tris pH 9.5, 2.5% 4 M NaCl and 0.5% 1 M MgCl2. Same molecular weight markers were used in the gel electrophoresis. 2.8. Titer determination using ELISA ELISA was done in the samples of cobra venom using antiserum produced against and individual proteins. The flat-bottomed ELISA plates were coated with venom sample in PBS for overnight at 4  C. The plates were washed thoroughly with PBS and blocked with 2% BSA in PBS for 1 h at 37  C. Subsequently, the plates were washed thoroughly with PBS and incubated with antiserum raised against cobra venom and individual venom proteins at 37  C for 2 h. The plates were washed with PBS/T and PBS three times each for 2 min and further incubated with 100 ml of rabbit anti-mouse IgG conjugated with alkaline phosphatase for 1 h. The plates were washed with PB/T and PBS three times each for 2 min and developed with the substrate p-nitrophenyl phosphate in substrate buffer. The optical density was measured at 405 nm using an automated ELISA reader and the titers were determined [16]. 2.9. Median lethal dose (LD50) determination The lethal toxicity was determined in male Swiss strain mice. Groups of six animals were injected i.p. with 0.5 ml of 0.85% NaCl containing increasing concentrations of cobra venom by the

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C. Venkatesan et al. / Biologicals 42 (2014) 8e21 Table 1 Antibody titers of whole venom antiserum and individual venom proteins antisera of cobra venom.a Groups

Antibody dilution

I II III IV V VI VII VIII Whole venom

5,12,000 No reaction 2,56,000 16,000 No reaction 1,28,000 1,28,000 1,28,000 1,28,000

Table 3 Percentage survival of mice at 24 h after injection of pre-incubated venom (25 mg/ mouse) with different volumes of various antisera raised against different proteins of cobra venom. Values are mean  SE (n ¼ 18). Different types of antisera

Mixturea (Venom in PBS þ antiserum) (ml/animal)

Normal serum

400 300 200 100 400 300 200 100 400 300 200 100 400 300 200 100 400 300 200 100 400 300 200 100 400 300 200 100 400 300 200 100 500

Antiserum of 14 kDa protein

a

The antibody titers were accessed by ELISA using whole cobra venom or individual venom protein as an antigen. Antibody titer corresponds to the maximal dilution of the serum resulting in OD 405 nm values higher than 0.100.

method [17]. The different concentrations of venom used for determination of LD50 value for the route was 6.25, 7.81, 9.76, 12.20, 15.25 and 19.90 mg/animal. The dose was killed 50% of animals within 24 h after determined by SpearmaneKarber method [18].

Antiserum of 29 kDa protein

Antiserum of 45 kDa protein

Antiserum of 65 kDa protein

2.10. In vitro cytotoxicity of cobra venom on Vero cell line 2.10.1. MTT assay (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) MTT assay as described by Mosmann [19] is based on inhibition by chemical injury of the reduction of soluble yellow MTT tetrazolium salt to a blue insoluble MTT formazan product by mitochondrial succinic dehydrogenase. The healthy cells were seeded in 96 wells microtiter plates at a concentration of 6  104 cells per well, and incubated at 37  C, in CO2 incubator with 5% CO2. After the formation of monolayer, the culture medium was removed and fresh medium with different concentrations of whole venom or its individual proteins were added. The concentrations used were 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg/ml. The experiments were carried out in triplicates. After 24 h exposure, the test medium was replaced by 20 ml of 0.5% MTT (Sigma, St. Louis) in PBS. MTT stock solution was prepared by filtering through a 0.22 mm filter to sterilize and remove the insoluble residues. After staining for 4 h at 20  C, the staining solution was carefully removed by aspiration and the cells were rinsed twice with PBS rapidly, and then 150 ml/well of DMSO or acidified isopropanol (100 ml of conc. hydrochloric acid in 100 ml of isopropanol) was added to solubilize the blue formazan crystals produced. The absorbance of each well was measured using a test wavelength at 570 nm and reference wavelength at 630 nm with a microplate reader. 2.10.2. Neutral red (NR) assay The method as described by Borenfreund and Puerner [20] and Schirmer [21] measures inhibition of cell growth, which is based on

Antiserum of 72 kDa protein

Antiserum of 99 kDa protein

Whole venom antiserum

Negative control (PBS only)

Percentage survival

þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 þ 100 ¼ 500 þ 200 ¼ 500 þ 300 ¼ 500 þ 400 ¼ 500 (ml/mouse)

00.00 00.00 00.00 00.00 00.00 33.30 55.53 66.64 00.00 11.07 27.73 38.87 00.00 5.53 22.17 27.73 00.00 5.53 22.17 38.87 00.00 5.53 22.17 38.87 00.00 22.17 44.43 66.64 00.00 16.60 83.30 100.00 100.00

                                

0.00 0.00 0.00 0.00 0.00 0.00 5.53 9.62 0.00 5.53 5.57 5.57 0.00 5.53 5.57 5.57 0.00 5.53 5.57 5.57 0.00 5.53 5.57 5.57 0.00 5.57 5.57 9.62 0.00 5.53 5.57 0.00 0.00

Mixture contained venom (25 mg/mouse) dissolved in PBS and antiserum in different volumes (100, 200, 300 and 400 ml), and made to 500 ml and incubated for 30 min at room temperature before injecting into mouse. a

the absorbance of the vital dye NR by living cells, but not by dead cells. The above-mentioned procedure was followed for NR assay. After 24 h exposure, the test medium was replaced by 200 ml NR solution containing 50 mg/ml of NR solution, which had been preincubated overnight at 20  C and filtered prior to use to remove fine precipitates of dye crystals. After in situ incubation for 3 h at 20  C, the plates were rinsed with warmed PBS, and the cells were destained with 200 ml de-staining solution (glacial acetic acid:96% ethanol:water, 1:50:49, by volume). After rapid agitation for 10 min at room temperature, the absorbance of the solution in each well was measured at 540 nm with a microplate reader.

Table 2 Cumulative percent mortality of mice injected with whole venom or different individual venom protein at different time intervals (n ¼ 18). The values are mean  SE. Treatments

Whole venom Band I Band II Band III Band IV Band V Band VI Band VII Band VIII PBS

Concentration (mg/mouse)

Cumulative percent mortality at different time intervals (h post-injection)

25 30 30 30 30 30 30 30 30 0

27.73 38.86 0.00 27.73 22.17 0.00 16.60 22.16 11.07 00.00

6

12          

5.56 5.56 0.00 5.56 5.57 0.00 0.00 5.57 0.00 0.00

61.07 55.50 0.00 33.30 44.43 0.00 22.17 16.73 22.07 0.00

18          

5.53 5.53 0.00 9.64 5.57 0.00 5.57 9.70 5.47 0.00

77.73 72.17 16.60 66.63 55.53 0.00 44.43 27.73 38.93 0.00

24          

5.57 5.57 0.00 9.6 5.53 0.00 5.57 5.57 5.53 0.00

100.00 88.86 22.17 77.73 66.63 0.00 61.07 55.53 61.10 0.00

         

0.00 5.57 5.57 5.57 9.61 0.00 5.53 5.53 11.10 0.00

C. Venkatesan et al. / Biologicals 42 (2014) 8e21 Table 4 Percentage survival of mice at 24 h after injection of pre-incubated venom (25 mg/ mouse) with different volumes of whole venom antiserum, commercial antiserum or cocktail antiserum.a Values are mean  SE (n ¼ 18). Different types of antisera

Mixtureb (venom in PBS þ antiserum) (ml/animal)

Whole venom antiserum

400 300 200 100 400 300 200 100 400 300 200 100 400 300 200 100

Cocktail antiseruma

Commercial antiserum

Normal serum

þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ

Percentage survival

100 ¼ 500 200 ¼ 500 300 ¼ 500 400 ¼ 500 100c ¼ 500 200d ¼ 500 300e ¼ 500 400f ¼ 500 100 ¼ 500 200 ¼ 500 300 ¼ 500 400 ¼ 500 100 ¼ 500 200 ¼ 500 300 ¼ 500 400 ¼ 500

00.00 16.60 83.30 100.00 00.00 83.30 100.00 100.00 00.00 11.07 83.30 100.00 00.00 00.00 00.00 00.00

               

0.00 5.53 5.57 0.00 0.00 5.57 0.00 0.00 0.00 5.53 5.57 0.00 0.00 0.00 0.00 0.00

a Cocktail antiserum was prepared by mixing of five antisera raised against venom proteins (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) at the ratio of 1:1:1:1:1. b Mixture contained venom (25 mg/mouse) dissolved in PBS and antiserum (whole venom antiserum, commercial antiserum or cocktail antiserum) in different volumes (100, 200, 300 and 400 ml), and made to 500 ml and incubated for 30 min at room temperature before injecting into mouse. c 20 ml from each antivenom (20  5)  100 ml. d 40 ml from each antivenom (40  5)  200 ml. e 60 ml from each antivenom (6  5)  300 ml. f 80 ml from each antivenom (80  5)  400 ml.

2.11. Neutralization of lethal activity of N. naja venom 2.11.1. Determination of median effective dose (ED50) of rabbit serum against N. naja venom The cocktail antiserum was prepared by mixing of these five antisera (14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa) at the ratio of 1:1:1:1:1 were selected based on antibody titer, Western blot analysis and neutralization efficiency. The venom at the dose of 2 LD50 was mixed with varying amounts of rabbit antivenom (100, 200, 300 and 400 ml) made up to 500 ml where necessary with normal saline solution or cocktail antisera prepared from different antisera raised against different venom proteins of cobra. The mixtures were incubated at 37  C for 30 min before injection. Eleven groups of mice were used in the experiment. The positive control group was injected with the mixture normal serum of rabbit and N. naja venom (25 mg/animal). The mice in the negative control group were injected with PBS only. The mice in the other groups were given a mixture of N. naja venom (25 mg/mouse) and different types of antisera at different volumes as mentioned in Tables 6 and 7. Intraperitoneal route was followed in all the experiments. The numbers of surviving animals were recorded 24 h after injection and the median survival analysis was determined by SpearmaneKarber method with ED50 expressed as ml serum/mg venom. The experiment was conducted in triplicate.

11

2.11.2. Intraperitoneal administration of antiserum at 0, 1, 2, 4, 6, 8 and 10 h after injection of N. naja venom Three sets of experiments were conducted to study the efficacy of whole venom antiserum, cocktail antiserum and commercial antiserum to neutralize cobra venom in mice. Each set consisted of eight groups of mice (six per group). In the first set, the mice of Group I was injected (i.p.) a lethal dose (25 mg/animal) of N. naja venom and served as a positive control. In Group II, the mice were administered N. naja venom and whole venom antiserum (400 ml/mouse, i.p.) simultaneously through i.p. In Groups III, IV, V, VI, VII, VIII and IX the mice were injected N. naja venom (25 mg/mouse, i.p.). The mice in Group III were administered whole venom antiserum (400 ml/mouse, i.p.) 1 h after venom injection, Group IV (2 h), Group V (4 h), Group VI (6 h), Group VII (8 h) and Group VIII (10 h). The mice of Group IX were given PBS alone and served as a negative control. The number of surviving mice was recorded 24 h after injection of antiserum. The experiment was conducted in triplicate. The volume of cocktail antiserum and commercial antiserum used in this experiment was 300 ml and 400 ml per mouse. 2.12. Neutralization of cytotoxicity The efficacy of whole venom antiserum, cocktail antiserum and commercial antiserum to neutralize the cytotoxicity of cobra venom was tested in Vero cell line using MTT assay and Neutral red assay [19]. The healthy cells were seeded in 96 wells microtiter plates at a concentration of 6  104 cells per well in 100 ml medium, and incubated at 37  C, in CO2 incubator with 5% CO2 for 24 h. The cells were exposed to different concentrations (5 mge10 mg) of cobra venom. The cobra venom (5 mg) was mixed with different dilutions (20, 30 and 40 ml, and made up to 100 ml) and incubated for 1 h at 37  C. After incubation, the mixture was added to each well of microtitre plates and incubated for 24 h. The experiments were conducted in triplicates. After 24 h exposure, MTT assay and Neutral red assay were carried out as mentioned above to assess the cell survival. The results were analyzed using ANOVA. 2.13. Histopathological analysis For histological investigation, organs such as heart, liver, lungs, kidney, spleen and brain were dissected out from N. naja venom injected mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum and fixed in neutral buffered formalin fixative (10% formalin). After fixation, the tissue samples were processed for sectioning. The tissues were washed several times in 70% ethanol and then dehydrated by washing in ascending grades of ethanol before clearing with xylene. The tissues, after clearing, were left in a mixture of xylene and paraffin wax (approximately 1:1) at room temperature overnight. Before embedding, the tissues were impregnated in three changes of paraffin wax with ceresin of 58e60  C melting point for 1 h each. The transverse sections were cut at 5e7 mm thickness using a manual rotatory microtome. After deparaffinising in xylene,

Table 5 Neutralizing potency of whole venom antiserum, cocktail antiserum and commercial antiserum against lethal effect of Naja naja venom.a The values are mean  SE. Serial no.

Types of antisera

ED50 (ml/mouse)b (upper and lower limits) mean  SE

ED100 (ml/mouse)c (upper and lower limits) mean  SE

1 2 3

Whole venom antiserum (ml/mouse) Cocktail antiserum (ml/mouse) Commercial antiserum (ml/mouse)

ð130:13162:70 : ::::: :::::::::162:70 203:20Þ ð121:10137:77 : ::::: ::::::::137:77 157:40Þ ð181:43206:63 : ::::: ::::::::206:63 235:87Þ

ð280:61:::::: ::::::::365:60 320:30Þ ð224:71256:41 : ::::: ::::::::256:41 292:50Þ ð351:21:::::: ::::::::400:52 456:91Þ

a b c

Experiments performed by pre-incubating venom (25 mg per mouse) and various concentrations of different types of antisera raised against cobra venom. Effective dose 50% (ED50) is the volume of antiserum that protects 50% of mice population against lethal effect of venom. Effective dose 100% (ED100) is the volume of antiserum that protects 100% of mice population against lethal effect of venom.

12

C. Venkatesan et al. / Biologicals 42 (2014) 8e21

Table 6 Percentage survival of envenomated mice treated with different types of antisera at different time intervals (h) after injection of cobra venom. (n ¼ 18). The values are means  SE. Antiserum type

Whole venom antiserum (400 ml/animal) Cocktail antiserum (300 ml/animal) Commercial antiserum (400 ml/animal) Positive control (PBS and venom) Negative control (PBS only) a

Concentration of venom (mg/mouse) used

Percentage of survival at 24 h after administration of the respective antiserum 0 ha

1 ha

2 ha

4 ha

25

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

83.32  5.57

83.32  5.57

0.00  0.00

25

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

83.32  5.57

0.00  0.00

25

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

83.32  5.57

0.00  0.00

25

0.00  0.00

0.00  0.00

0.00  0.00

0.00  0.00

0.00  0.00

0.00  0.00

0.00  0.00

0

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

100.00  0.00

00.00  0.00

6 ha

8 ha

10 ha

Administration of antiserum at 0, 1, 2, 4, 6, 8 and 10 h after injection of cobra venom.

the sections were hydrated through graded series of alcohol up to 70% and stained with Harris alum hematoxylin and counterstained with 1% alcoholic eosin [22] and then mounted with glass cover slip in DPX mount through xylene and observed under the light microscope.

2.14. Statistical analysis Data are expressed as mean  SE. The Mann Whitney nonparametric tests and parametric student’s t test were used for tests of significance of differences between groups. A probability of less than 0.05 was accepted as significant. Statistical calculations were performed using SPSS (version 9) software.

Fig. 1. SDS-PAGE showing the protein pattern of Indian Spectacled cobra (Naja naja) venom. Lane M e Marker; Lanes 1e12. Naja naja venom.

3. Results

3.2. Western blot analysis

3.1. Protein concentration and SDS-PAGE analysis of cobra (N. naja naja) venom

Antiserum was raised individually against different eluted proteins (bands I, II, III, IV, V, VI, VII and VIII) of cobra venom and against whole venom of cobra. The concentration of protein in the samples eluted from the SDS-PAGE corresponding to bands I, II, III, IV, V, VI, VII and VIII was 23.57  0.61, 32.26  0.56, 21.23  0.55, 23.24  0.48, 16.68  0.36, 18.24  0.54, 35.35  56 and 28.24  0.61 (mg/ml), respectively. The production of antisera was confirmed by Western blot analysis (Fig. 2). The results of Western blot analysis showed the appearance of six bands when the whole venom was treated with antiserum raised against whole venom. The results of Western blot confirm the production of antiserum raised against the venom protein bands I, III, IV, VI, VII and VIII (clear bands were not observed in the case of bands II and V).

The concentration of protein in the venom of Spectacled cobra was 62.56  0.59 mg/ml. The protein profile of cobra venom was studied by SDS-PAGE. Fig. 1 shows the protein pattern of cobra venom. Eight protein bands (six major bands and two minor bands) were observed on SDS-PAGE in reducing condition after staining with Coomassie Brilliant Blue. The approximate molecular weight of protein bands I, II, III, IV, V, VI, VII and VIII calculated based on the standard protein markers such as 14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa and 99 kDa respectively (Fig. 1).

Table 7 Percentage of cell viability in Vero cell line treated with different concentrations of whole cobra venom or individual cobra venom proteins determined by MTT assay after 24 h of exposure. The values are mean  SE. Groups

Concentration of venoms (mg) 1

Wv I II III IV V VI VII VIII N

92.43 93.18 98.44 92.87 94.81 98.44 90.64 90.67 92.35 98.44

2          

1.64 0.96 0.18 1.20 0.64 0.18 0.34 0.34 0.17 0.18

80.94 66.01 98.44 72.53 91.08 97.45 80.94 80.94 78.53 98.44

3          

0.35 1.31 0.18 0.77 0.64 0.16 0.35 0.35 0.33 0.18

71.73 49.60 98.44 59.27 81.25 96.54 70.40 71.73 55.63 98.44

4          

1.72 2.21 0.18 0.16 0.33 0.19 0.00 0.99 0.33 0.18

Wv e whole venom of cobra, N e normal (PBS only).

64.35 26.34 97.55 38.48 77.33 96.44 63.35 64.35 41.0.43 98.44

5          

0.49 1.56 0.16 0.16 1.06 0.19 0.45 0.49 0.11 0.18

49.74 11.69 95.24 20.46 70.25 95.24 52.37 49.74 30.38 98.44

6          

0.31 0.95 0.16 0.78 0.00 0.11 0.66 0.31 0.13 0.18

33.24 9.33 93.24 15.92 67.31 95.54 43.41 33.24 25.67 98.44

7          

0.66 0.57 0.21 0.28 0.99 0.19 1.03 0.66 0.14 0.18

28.79 8.98 93.18 11.50 58.79 94.34 32.54 28.79 19.94 98.44

8          

0.29 0.62 0.96 0.02 0.72 0.12 0.68 0.29 0.32 0.18

21.40 6.04 93.18 10.34 49.10 93.52 26.17 21.79 18.40 98.44

9          

0.27 0.78 0.96 0.87 0.27 0.21 0.27 0.27 0.69 0.18

8.38 2.59 80.94 9.87 27.87 92.44 18.05 8.38 18.87 98.44

10          

0.33 0.31 0.35 0.33 1.11 0.16 0.35 0.10 0.33 0.18

4.02 1.02 71.73 9.35 21.25 91.34 14.10 4.02 15.36 98.44

         

0.77 0.78 1.72 0.78 0.63 0.10 0.34 0.77 0.18 0.18

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Table 9 Percentage survival of cells after exposing to venom or venom neutralized with different dilutions of various types of antisera using MTT assay after 24 h of exposure. The values are mean  SE. Treatmentsa

Percentage of survival

Whole venom antiserum (ml) 20 30 40 Commercial antiserum (ml) 20 30 40 Cocktail antiserum (ml) 20 30 40 Venom only (5 mg)

Fig. 2. Confirmation of production of antisera against whole venom and individual proteins of cobra venom in rabbit by Western blot analysis. Lane M e Marker; Lane 1 e Whole venom; Lane 2 e 14 kDa; Lane 3 e 29 kDa; Lane 4 e 45 kDa; Lane 5 e 65 kDa: Lane 6 e 72 kDa; Lane 7 e 99 kDa.

13.75  4.10 48.75  7.60 98.75  0.75 22.10  0.99 52.50  4.04 91.08  1.71 27.50 58.06 97.90 13.21

   

2.53 3.46 1.79 1.38

a Neutralization of cobra venom (5 mg) with different dilution of whole venom antiserum, cocktail antiserum or commercial antiserum.

3.3. Immunization and antibody response

3.6. Determination of median effective dose (ED50)

The whole cobra venom was assessed by ELISA for immunization. The antibody levels against whole venom were statistically significant in immunized rabbits when compared to negative controls to the PBS (Table 1).

The antiserum raised against whole venom protected all envenomated mice at the volume of 400 ml per mouse whereas at the volume of 300 ml per mouse, 83.3% survival of envenomated mice was observed. The antisera raised against different venom proteins showed different survival rates and none of the antisera showed 100% survival in envenomated mice. The highest survival (66.63%) was observed in the envenomated mice treated with the antiserum raised against venom protein 14 kDa or 99 kDa whereas lowest survival was observed in the envenomated mice treated with antiserum raised against the venom protein of 45 kDa. The cocktail antiserum was prepared by mixing of antiserum raised against 14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa at the ratio of 1:1:1:1:1 and tested its efficacy to protect the envenomated mice (Tables 3 and 4). All the envenomated mice treated with cocktail antiserum at the volume of 300 or 400 ml per mouse survived without any mortality whereas 83.3% survival and 100% mortality were observed in envenomated mice treated with 200 and 100 ml per mouse of cocktail antiserum, respectively. The ED50 and ED100 values of whole venom antiserum, commercial antiserum and cocktail antiserum were determined and the results are given in Table 5. The ED50 for the antiserum for whole venom, cocktail antiserum and commercial antiserum was found to be 162.7 ml per mouse, 137.77 ml per mouse and 206.63 ml per mouse, respectively.

3.4. Determination of LD50 The LD50 value of cobra venom was determined in the mice model by different routes. For intraperitoneal route the LD50 was found to be 13.73 mg/mouse. The upper and lower limits were 2.03 and 15.55 mg/mouse. 3.5. In vivo toxicity in mice The mice were injected with whole venom or individual proteins of cobra venom to determine the toxicity as well as 100% mortality within 24 h p.i. The results of in vivo test revealed that a dose of 25 mg/animal caused 100% mortality after 24 h p.i. in mice injected through i.p. route. The mortality rate varied in mice injected with different individual proteins of venom separately. The band I caused 88% mortality and the remaining bands caused mortality which ranged from 55 to 77% at 24 h p.i. (Table 2). In the envenomated mice, paralysis started at the injected leg and then whole body became paralyzed at moribund stage. The animal could not stand, breathing became difficult and airway exudation was observed. The animals died as a result of paralysis and airway block within 14 h.

3.7. Intraperitoneal administration of antiserum at 0, 1, 2, 4, 6, 8 and 10 h after injection of cobra venom 100% survival was observed in envenomated mice treated with whole venom antiserum at the dose of 400 ml/mouse at 0, 1, 2

Table 8 Percentage of cell viability in Vero cell line treated with different concentrations of whole cobra venom or individual cobra venom proteins determined by Neutral red (NR) assay after 24 h of exposure. The values are mean  SE. Groups

Concentration of venoms (mg)

Wv I II III IV V VI VII VIII N

92.43 92.43 98.44 92.35 92.87 98.44 92.43 93.18 92.87 98.44

1

2          

1.64 1.64 0.18 0.17 1.20 0.18 1.64 0.96 1.20 0.18

80.94 80.94 98.44 78.53 72.53 98.44 80.94 66.01 72.53 98.44

3          

0.35 0.35 0.18 0.33 0.77 0.18 0.35 1.31 0.77 0.18

71.73 71.73 98.44 55.63 59.27 98.44 71.73 49.60 59.27 98.44

4          

1.72 1.72 0.18 0.33 0.16 0.18 1.72 2.21 0.16 0.18

Wv e whole venom of cobra, N e normal (PBS only).

64.35 64.35 97.55 41.43 38.48 98.44 64.35 26.34 38.48 98.44

5          

0.49 0.49 0.16 0.11 0.16 0.18 0.49 1.56 0.16 0.18

49.74 49.74 95.24 30.38 20.46 95.24 49.74 11.69 20.46 98.44

6          

0.31 0.31 0.16 0.13 0.78 0.16 0.31 0.95 0.78 0.18

33.24 33.24 93.24 25.67 15.92 93.18 33.24 9.33 15.92 98.44

7          

0.66 0.66 0.21 0.14 0.28 0.96 0.66 0.57 0.28 0.18

28.79 28.79 93.18 19.94 11.50 93.18 28.79 8.98 11.50 98.44

8          

0.29 0.29 0.96 0.32 0.02 0.96 0.29 0.62 0.02 0.18

21.40 21.40 93.18 18.40 10.34 93.18 21.40 6.04 10.34 98.44

9          

0.27 0.27 0.96 0.69 0.87 0.96 0.27 0.78 0.87 0.18

8.38 8.38 80.94 18.87 9.87 93.18 8.38 2.59 9.87 98.44

10          

0.33 0.33 0.35 0.33 0.33 0.96 0.33 0.31 0.33 0.18

4.02 4.02 71.73 15.36 9.35 93.18 4.02 1.02 9.35 98.44

         

0.77 0.77 1.72 0.18 0.78 0.96 0.77 0.78 0.78 0.18

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C. Venkatesan et al. / Biologicals 42 (2014) 8e21 Table 10 Percentage survival of cells after exposing to venom or venom neutralized with different dilutions of various types of antisera using Neutral red (NR) assay after 24 h of exposure. The values are mean  SE. Treatmentsa Whole venom antiserum (ml) 20 30 40 Commercial antiserum (ml) 20 30 40 Cocktail antiserum (ml) 20 30 40 Venom only (5 mg)

Percentage survival 16.15  0.99 26.68  5.76 96.11  2.45 16.10  0.99 34.50  4.04 94.66  4.71 30.48 57.03 96.13 13.21

   

5.33 6.08 1.12 1.38

a Neutralization of cobra venom (5 mg) with different dilution of whole venom antiserum, cocktail antiserum or commercial antiserum.

and 4 h after injection of cobra venom and 83.32% survival in mice administered the antiserum at 6 and 8 h after venom injection. Similar results were observed in the mice treated with commercial antiserum but the dose was 400 ml/mouse and 100% survival was observed in the case of mice treated with cocktail antiserum (300 ml per mouse) at 0, 1, 2, 4 and 6 h after injection of venom (Table 6). 3.8. In vitro cytotoxicity of cobra venom on Vero cell line The cytotoxicity of the whole venom or individual venom proteins of N. naja venom with various concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 mg) was tested using Vero cell line and cell survival was determined using MTT assay and NR assay. The results are given in Tables 7 and 8. The result of MTT and NR assay showed that whole cobra venom or individual venom proteins induced cell death in a dose dependent manner. The whole venom and protein bands I, III, VI, VII and VIII caused more than 95% cell death in high

Fig. 3. Cytotoxicity of cobra venom and cobra venom neutralized with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal Vero cells; B e Vero cells treated with the mixture of cobra venom and cocktail antiserum; C e Vero cells treated with the mixture of cobra venom and whole venom antiserum; D e Vero cells treated with the mixture of cobra venom and commercial antiserum; E e Vero cells treated with cobra venom only.

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Fig. 4. Histological changes in the kidney of envenomated mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal mice; B e Envenomated mice, note the glomerular congestion and hemorrhagic regions (arrow). C e Envenomated mice treated with cocktail antiserum with normal structure. D e Envenomated mice treated whole venom antiserum and no any change in the tissue. E e Envenomated mice treated with commercial antiserum and normal feature was observed in the kidney.

concentrations whereas lower concentrations caused less than 50% cell death (Tables 7 and 8). 3.9. Neutralization of cytotoxicity of cobra venom in Vero cells The cobra venom was found to be highly toxic to Vero cells from a concentration of 5 mg onwards (Tables 7 and 8). The venom at the concentration of 5 mg was neutralized with various volumes (20, 30 and 40 ml) of whole venom antiserum, cocktail antisera or commercial antiserum (Tables 9 and 10). The survival of cells was measured by MTT and NR assays. The survival of cells exposed to venom neutralized with high concentration of antiserum of different preparations was found to be significantly higher than the control groups as well as venom treated with low concentration of antiserum. The percentage survival of cells measured by MTT assay was estimated about 13.75, 48.75 and 98.75 in venom neutralized with whole venom antiserum at the concentration of 20, 30 and 40 ml, respectively. The percentage survival of cells measured by MTT assay was estimated about 22.10, 52.50 and 91.08 in venom neutralized with commercial antiserum at the concentration of 20, 30 and 40 ml, respectively. The percentage survival of cells measured by MTT assay was estimated about 27.50, 58.06 and 97.90 in venom neutralized with cocktail antiserum at the concentration

of 20, 30 and 40 ml, respectively. In the case of venom alone, the percentage cell survival was estimated about 13.21 after 24 h exposure (Table 10). The percentage survival of cells treated with venom and neutralized venom was also measured using NR assay (Table 10). The percentage survival of cells measured by NR assay was estimated about 16.15, 26.68 and 96.11 in venom neutralized with whole venom antiserum at the concentration of 20, 30 and 40 ml, respectively. The percentage survival of cells measured by NR assay was estimated about 16.10, 34.50 and 94.66 in venom neutralized with commercial antiserum at the concentration of 20, 30 and 40 ml, respectively. The percentage survival of cells measured by NR assay was estimated about 30.48, 57.03 and 96.13 in venom neutralized with cocktail antiserum at the concentration of 20, 30 and 40 ml, respectively. In the case of venom alone, the percentage cell survival was estimated about 11.31 after 24 h exposure (Fig. 3). 3.10. Histological investigation Histological changes were observed in different organs (heart, liver, lungs, kidney, spleen and brain) of envenomated mice and treated with cocktail antiserum, whole venom antiserum or commercial antiserum after 48 h of treatment and the results are

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Fig. 5. Histological changes in the liver tissue of envenomated mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal mice; B e Envenomated mice, note the congestion of blood vessels (arrow). C e Envenomated mice treated with cocktail antiserum. D e Envenomated mice treated with whole venom antiserum. E e Envenomated mice treated with commercial antiserum. No changes were observed in CeE.

shown in Figs. 4e9. Tissue sections of different organs of normal mice injected with PBS showed a normal histological pattern (Figs. 4Ae9A). Significant histological changes were observed in all the organs of envenomated mice (Figs. 4Be9B). Histological observations in kidney tissue of envenomated mice at moribund stage showed the glomerular congestions, vacuolar degeneration of tubular cells and hemorrhagic regions (Fig. 4B). Microscopic examination of liver tissue of envenomated mice indicates the disturbances of circulation in the liver tissue with marked protein dystrophy in hepatocytes. Degenerated hepatocytes were also observed in different regions of liver tissue (Fig. 5B). Microscopic examination of lung tissue of envenomated mice showed infiltration of erythrocytes within bronchus alveoli and thickening of partitions between alveoli (Fig. 6B). Interalveolar partitions were torn in some places of lung tissue. Histological examination of heart sections of envenomated mice revealed a disruption in the striations of the muscle with hemorrhagic areas (Fig. 7B). Several blood vessels were found to be congested and capillaries were filled with erythrocytes. Histological observation of spleen of envenomated mice revealed the congestion of red pulp

(Fig. 8B). Cells with autophagy and poly-nucleate cells were observed in the spleen tissue of envenomated mice at moribund stage. Microscopic examination of brain tissue of envenomated mice showed no significant changes in the cells except some marked infiltration of leukocytes (Fig. 9B). Envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum did not show any observable changes in kidney (Fig. 4CeF), liver (Fig. 5CeE), lung (Fig. 6CeE), heart (Fig. 7CeE), spleen (Fig. 8CeE) and brain (Fig. 9CeE) after 48 h of treatment. 4. Discussion The protein pattern of venoms of the medically important Indian snake (Spectacled cobra) was studied by SDS-PAGE. Eight protein bands with molecular weights of 14 kDa, 24 kDa, 29 kDa, 45 kDa, 48 kDa, 65 kDa, 72 kDa, and 99 kDa were observed on SDS-PAGE in the venom of N. naja. Similar type of protein banding pattern was observed by many workers [10,23e25]. Mendoza and Bhatti [23] observed seven protein bands in SDSPAGE and twelve bands in non-SDS electrophoresis. SDS-PAGE analysis was carried out on Indian cobra venom obtained from

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Fig. 6. Histological changes in the lung tissue of envenomated mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal mice; B e Envenomated mice, note the infiltration of erythrocytes and thickening of partitions between alveoli (arrow). C e Envenomated mice treated with cocktail antiserum with normal structure. D e Envenomated mice treated with whole venom antiserum. Observed infiltration of erythrocytes. E e Envenomated mice treated with commercial antiserum with infiltration of erythrocytes.

three different geographical regions and the results revealed the presence of seven bands and significant variation in the protein constituents of the three regional venoms [25]. Cobra venom subjected to SDS-PAGE analysis indicated the presence of prominent protein components with molecular weights of 10 kDa, 20 kDa, 24 kDa, 55 kDa, 105 kDa and 110 kDa [10]. Immunotherapy using polyvalent antivenom raised in higher animals is the only effective treatment against snake venom poisoning. Moreover, although the advantages of polyvalent antivenom are quite obvious, there is still a belief that polyvalent antivenoms are less effective and cause higher incidence of adverse reactions when compared to monovalent antivenoms [26,27]. Hence, an alternate technology should be explored to produce antivenom. In the present study, an attempt was made to make use of a cocktail antiserum prepared by mixing of antisera raised against individual venom proteins of cobra venom. Highly potent antivenom against Thai cobra venom was produced in horse using different adjuvants and the results showed that the peak ELISA titer rose slowly and reached high levels after 18th week when bentonite was used whereas the ELISA titer rose very rapidly and reached high levels by 4th week when complete

Freund’s adjuvant (CFA) was used [28]. In the present study, the CFA was used to raise the antiserum against whole venom or individual venom proteins and the ELISA results showed high level antibody titer values for whole venom as well as some of the venom proteins [29]. Chotwiwatthanakun et al. (2001) produced potent polyvalent antivenom against Thai cobra and King cobra venom using low volume of venom with multiple-site immunization protocol as in the present study [30]. In the present study, the toxicity of cobra whole venom and individual venom proteins was tested in vivo using mice and in vitro using Vero cell line. The whole venom was given to mice through four routes namely intraperitoneal, intramuscular, intravenous and sub-cutaneous to determine the LD50 value for cobra venom. The LD50 value of cobra venom in mice weighing 20e22 g for intraperitoneal route was determined as 13.73 mg/mouse [31]. The LD50 values of cobra venom through different routes in the present study coincided with values reported by many workers [2,32e37]. Shashidharamurthy et al. (2002) conducted toxicity experiments with cobra venom collected from different regions of India in mice through intraperitoneal route and calculated the LD50 value as

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Fig. 7. Histological changes in the heart tissue of envenomated mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal mice; B e Envenomated mice, note the disruption in muscle fibers with hemorrhagic regions (arrow). C e Envenomated mice treated with cocktail antiserum. D e Envenomated mice treated whole venom antiserum. E e Envenomated mice treated with commercial antiserum. Normal texture was observed in the heart tissue in treated envenomated mice.

14 mg/mouse for eastern, 24 mg/mouse for western and 40 mg/ mouse for southern venoms of cobra [24]. Osipov et al. [38] have studied the toxicity of Thailand and Middle-Asian cobra venoms and their isolated components in cockroach using intra-abdominal injection and the results revealed that most of the components are not toxic for cockroaches up to 15 nmol/g but are toxic to mice. Antisera in different dilutions such as 100, 200, 300 and 400 ml were used to neutralize the cobra venom at the concentration of 25 mg/mouse. The antiserum raised against whole venom showed 100% survival in mice injected with the mixture of 400 ml of antiserum and 25 mg of venom/mouse whereas the mortality was found to be 100, 83.4 and 16.7% in mice injected with the mixture of 100 ml of antiserum and 25 mg of venom/ mouse, 200 ml of antiserum and 25 mg/mouse and 300 ml of antiserum and 25 mg of venom/mouse, respectively. Based on the results of Western blot, antibody titer and neutralization studies, the antisera raised against 14 kDa, 29 kDa, 65 kDa, 72 kDa and 99 kDa were selected to prepare cocktail antiserum for neutralization and other studies.

The cocktail antiserum was used in the present study to neutralize the cobra venom for developing an alternate technology for the production of antivenom using monoclonal antibodies for specific antigenic venom proteins. The use of cocktail antiserum to neutralize the cobra venom will form the basis for developing antivenom using monoclonal antibodies. Many workers have tried to develop alternate technology for production of antivenom against different snake venoms due to various reasons particularly the adverse side effects such as anaphylactoid reactions and serum sickness [36,37,39e41]. All these workers have tried to generate polyclonal antibodies in chicken egg against venoms of cobra, krait and vipers. Almeida [39] reported that polyclonal antibodies raised in chicken egg were found to be capable of recognizing, combining with and neutralizing the toxic and lethal components of Bothrops and Crotalus venoms. The efficiency of cocktail antiserum to neutralize the cobra venom was tested by two strategies as mentioned by Shashidharamurthy and Kemparaju [35]. In the first strategy, the venom was mixed with cocktail antiserum and incubated for 30 min prior to injecting into mice. The neutralization efficiency was significantly

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Fig. 8. Histological changes in the spleen of envenomated mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal mice; B e Envenomated mice, note the congestion of red pulp (arrow). C e Envenomated mice treated with cocktail antiserum with normal structure. D e Envenomated mice treated whole venom antiserum and no any change in the tissue. E e Envenomated mice treated with commercial antiserum and normal feature was observed in spleen.

higher when compared to whole venom antiserum and commercial antivenom. The percentage of survival was found to be significantly higher in mice treated with cocktail antiserum in comparison with mice treated with whole venom antiserum or commercial antiserum. The ED50 value for cocktail antiserum, whole venom antiserum and commercial antiserum was calculated as 137.77, 162.7 and 206.63 ml/mouse, respectively. In the second strategy, the venom and antivenom were injected separately into experimental mice. In addition the envenomated mice were injected with antivenom at different time intervals after injecting the venom. Table 9 shows the influence of time intervals between envenomation and antivenom administration on the efficacy of cocktail antiserum, whole venom antiserum and commercial antiserum. The results showed that the cocktail antiserum or commercial antiserum protected all envenomated mice which were treated with antisera 6 h after injection of venom whereas 83.32% survival was observed in envenomated mice treated with whole venom antiserum 6 h after venom injection. All the envenomated mice died when treated with different types of antisera after 8 h of venom injection. Our observation agrees with the results of Chaves [42]. The results of the present study agree with previous observations made by different workers [30,35,42]. In the pre-incubation stage, the venom was totally neutralized with antivenom and caused least damage whereas in second strategy, when antivenom was administered after venom injection, neutralization was only partial and tissue damage developed very fast. Based on the results of the present

study, it is recommended that the cocktail antiserum should be given to envenomated mice before 6 h before venom injection to avoid tissue damage due to venom as recommended by previous workers [35,42,43]. The cytotoxicity of whole cobra venom and individual venom proteins was also tested in vitro using Vero cell line by MTT and NR assays. The cell line was exposed to different concentrations of whole venom or individual venom proteins and the results showed that the venom at the concentration of 3 mg and above caused significant mortality in the cell line. The individual venom proteins of 14 kDa, 29 kDa, 45 kDa, 65 kDa, 72 kDa and 99 kDa caused significant cytotoxicity in Vero cell line whereas 24 and 48 did not show significant cytotoxicity in the cell line. Both in vivo and in vitro studies revealed the toxicity of whole venom and individual venom proteins of 14 kDa, 29 kDa, 45 kDa, 65 kDa, 72 kDa and 99 kDa in mice as well as in Vero cell line. Based on these studies, 25 mg/mouse and 5 mg/well were selected for neutralization studies by in vivo and in vitro, respectively, using mice and Vero cell line [44]. The efficiency of cocktail antiserum, whole venom antiserum and commercial antiserum to neutralize the cobra venom was tested in vitro using Vero cell line by MTT assay. The results indicate that the venom treated with cocktail antiserum, whole venom antiserum and commercial antiserum at the concentration of 40 ml/ well protected the Vero cells against cytotoxic effects of cobra venom and showed 97.9, 98.75 and 91.08% cell survival,

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Fig. 9. Histological changes in the brain of envenomated mice and envenomated mice treated with cocktail antiserum, whole venom antiserum or commercial antiserum. A e Normal mice; B e Envenomated mice. C e Envenomated mice treated with cocktail antiserum. D e Envenomated mice treated whole venom antiserum. E  Envenomated mice treated with commercial antiserum. No significant changes were observed in envenomated mice and treated envenomated mice.

respectively. Both in vitro and in vivo studies showed that cocktail antiserum gave significant protection against cytotoxic effects and lethality of cobra venom in Vero cells and mice, respectively as whole venom or commercial antiserum. Significant histological changes such as infiltration of erythrocytes and congestion of blood vessels were observed in the lung tissue as observed by Ali et al. (2000); Aznaurian and Amiryan (2006) [45,46]. Ali et al. (2000) have concluded that the mechanism for lung injury was not due to the direct action of the venom or its components, but probably due to factors related to renal dysfunction due to envenomation. Light microscopic examination of the heart tissue sections of envenomated mice revealed a disruption in the striations of the muscles as observed by many workers [45,46]. This change may cause irregularities in contraction of atria and ventricles of the heart and rhythmical beating of heart, which in turn affects the circulation of blood in the body. Histological observation of liver tissue of envenomated mice revealed the congestion of blood vessels and capillaries. The obstruction of blood vessels may cause liver dysfunction. Based on the neutralization studies, the possibility of using monoclonal antibodies to treat snakebite is quite encouraging and the results of the present study on cobra venom could form the basis for developing monoclonal antibodies for various venoms.

5. Conclusion In conclusion, these data suggest that the present investigation has thrown up the possibility of developing alternate technologies for production of antivenom against cobra venom. The results obtained from various studies on use of cocktail antiserum in the present investigation can form the basis for developing antivenom using monoclonal antibodies for snakebite treatment in future. Acknowledgment The first author is recipient of a Young Scientist award from the Department of Science and Technology, Government of India and partially funded by Department Biotechnology (DBT), Government of India. The authors thank the management of C.Abdul Hakeem College for providing facilities to carry out this work. The secretory, Irula’s snake catcher’s co-operative society, Chennai for providing the snake venoms for this study. The authors are thankful to King Institute of Preventive Medicine for providing Vero cell line. References [1] Chippaux JP. Snake-bites: appraisal of the global situation. Bull World Health Organ 1998;76:515e24.

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