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Abstract

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect the venom of Indian cobra (Naja naja naja) in various tissues (brain, heart, lungs, liver, spleen, blood, kidneys, and tissue at the site of injection) of mice after cobra venom injected at different time intervals (0, 2, 4, 6, 8, and 12 h intervals up to 24 h). Whole venom antiserum or individual venom protein antiserum (14, 29, 65, 72, and 99 kDa) could recognize N. n. naja venom by Western blotting and ELISA, and antibody titer was also assayed by ELISA. Antiserum raised against cobra venom in rabbit significantly neutralized the toxicity of venom-injected mice at different time intervals after treatment. The assay could detect N. n. naja venom levels up to 2.5 ng/ml of tissue homogenate, and the venom was detected up to 24 h after venom injection. Venom was detected in brain, heart, lungs, liver, spleen, kidneys, tissue at the bite area, and blood. As observed in mice, tissue at the site of bite area showed the highest concentration of venom and the brain showed the least. Moderate amounts of venoms were found in liver, spleen, kidneys, heart, and lungs. Development of a simple, rapid, and species-specific diagnostic kit based on this ELISA technique useful to clinicians is discussed.

Keywords

ELISA antiserum cocktail antiserum cobra

Introduction

Snake bite is an occupational hazard, especially among the snake catchers, forest workers, and agriculturists in rural India. It is estimated that about 200,000 persons are bitten in India annually and about 15,000 are fatal¹ but hospital statistics include only about 10% of the total cases in the population.² The incidence is more in rural areas during the rainy season, particularly at nights. A total of about 216 species of snakes are found in India of which only 52 species are venomous. The four major venomous snakes chiefly responsible for the injuries and fatalities in India are Krait (Bungarus caeruleus), cobra (Naja naja naja), saw-scaled or carpet viper (Echis carinatus), and Russell’s viper (Daboia russelli russelli).^3,4 There is no rapid and dependable diagnostic test for the identification of species responsible for envenomation; hence, polyvalent antiserum raised against a mixture of venoms from four major Indian poisonous snakes is being used for treatment. Production of polyvalent serum is a long and complex process and is expensive.⁵ Although it is effective, the recovery is slow and large volumes are needed for the treatment of any particular snake bite. Further, the severe allergic reactions, serum sickness, and other side effects as a consequence of these treatments are of serious concern.^6,7 Establishment of identity of the species of snake inflicting the bite would facilitate administration of monovalent (species specific) antiserum for rapid and effective recovery with reduced side effects.

There are several reports on the detection of snake venoms from various parts of the world, and the techniques were extensively reviewed.^8

–11 In several respects, enzyme-linked immunosorbent assay (ELISA) is of more practical use than any other tests, and it can be readily modified into a kit for field use. To begin with, a mouse model was adapted to develop ELISA for the detection of cobra venom. This was owing to the low venom levels in tissue samples, which made it essential that the ELISA should be more sensitive. Also, it would be advantageous to decrease the assay time considerably in view of the fact that venom could be detected in the samples as fast as possible. In this study, we report the development of a sandwich ELISA for the detection of Indian cobra venom in necropsy specimens of mice.

Materials and methods

Venom and experimental animals

The lyophilized whole venoms of four major Indian venomous snakes B. caeruleus, N. n. naja, E. carinatus and D. r. russelli were obtained from Irula Snake Catchers’ Society (Chennai, Tamil Nadu, India) with proper permission (No.WL1/7/2006, dated November 13, 2006) and preserved in desiccator at 4°C for further use. It was dissolved in 0.85% saline and centrifuged at 2000 r/min for 10 min. The supernatant was collected and kept at 4°C until further use. Venom concentration was expressed in terms of dry weight. Swiss albino mice (weighing 18–22 g) and New Zealand white male rabbits (weighing 2 ± 3 kg) obtained from the Institute of Veterinary Preventive Medicine (Ranipet, Tamil Nadu, India) were used for the study. They were kept in animal cages with sawdust as bedding under conditions of 12 h: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. All 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, dated December 19, 2008).

Chemicals

Tween 20, bovine serum albumin (Sigma, St Louis, Missouri, USA), o-phenylenediamine (Aldrich, Milwaukee, Wisconsin, USA), sephacryl S-200, protein A sepharose CL-4B (Pharmacia, Uppsala, Sweden), rabbit anti-goat immunoglobulin G (IgG) and goat anti-rabbit IgG ± horse radish peroxidase (HRP) conjugates (Dako, Denmark), biotin ± N-hydroxysuccinimide ester (Vega Biochemicals, Tucson, Arizona, USA), HRP ± avidin conjugate (Vector Laboratories, Burlingame, California, USA), hydrogen peroxide (Glaxo Laboratories, Bombay, Maharashtra, India), and polystyrene 96-well microtiter plates, at bottom (Costar, Cambridge, Massachusetts, USA) were obtained from the manufacturers. All other chemicals and reagents used were of analytical grade.

SDS-PAGE analysis of snake venom

The proteins of the medically important cobra (N. n. naja) snake venoms were analyzed by 12% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE).^12,13 The venom samples were subjected to SDS-PAGE for protein patterns. The samples were mixed with Laemmli sample buffer (0.0625 M tris(hydroxymethyl)aminomethane (Tris)–hydrochloric acid (HCl); pH 6.8, 2% SDS, 10% glycerol, 5% 2–mercaptoethanol, and 0.001% 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 coelectrophoresed.

Electroelution of different proteins of N. n. naja venom

The major venom proteins were electroeluted by SDS-PAGE.¹⁴ A preparative SDS-PAGE was run with proteins of cobra venom. After the run, the gel was soaked in prechilled potassium chloride (0.4 M). The prominent venom protein bands were excised, and the gel slices were minced into small pieces (approximately1 mm) using a sterile razor blade. The gel pieces were transferred into a dialysis bag with Tris–ethylenediaminetetraacetic acid (EDTA; TE) buffer (10 mM Tris–HCl 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 was dialyzed and concentrated by a SpeedVac evaporator (Lark Innovative Fine Teknowledge, Chennai, Tamail Nadu, India). The purified venom proteins were estimated and confirmed on SDS-PAGE.

Production of antiserum

Male healthy New Zealand white rabbits (2–3 kg body mass) were chosen for the production of polyclonal antibodies. Detoxified cobra venom (200 μg/kg of body mass) was emulsified with equal volume of complete Freund’s adjuvant (CFA)¹⁵ and injected intramuscularly (i.m.) at multiple sites. First booster dose (200 μg/kg of body mass) was i.m. given along with Freund’s incomplete adjuvant , after 4 weeks of the first dose. After first booster dose, injections, that is, second and third booster doses (200 μg/kg of body mass), were administered i.m. along with Freund’s incomplete adjuvant at an interval of 2 weeks.¹⁶ Blood samples were collected from the marginal ear vein. After coagulation, the blood sample was centrifuged at 2000g for 10 min, and the serum was collected for the determination of antibody titer using ELISA and Western blot analysis.^17,18 The increase in antibody levels in both goat and rabbit were monitored by assaying sera samples, obtained 1 week after the booster dose by ELISA using the respective venom as coating antigen.

ELISA for the determination of anti-snake venom antibodies

Microtiter wells were coated with the four venoms (5 mg/ml) and incubated with serum samples diluted in phosphate-buffered saline (PBS), pH 7.4 ± 2% rabbit serum ± Tween-20 for 1 h at 37.8°C. Bound anti-snake venom antibodies were detected using goat anti-rabbit IgG ± HRP conjugate, and the enzymatic activity was measured with peroxide substrate solution.

Western blot analysis

Cobra venom (20 µg) was first fractionated by SDS-PAGE, as described above. The gel was placed in the electroblotting apparatus adjacent to nitrocellulose paper (NCP) in buffer, as described by Towbin et al.¹⁹ After transfer, the NCP was blocked for 1 h with 3% skimmed milk in PBS (20 nM sodium phosphate containing 0.9% sodium chloride (NaCl), pH 7.2). The NCP was washed in PBS for 5 min and then incubated with 1:10,000 dilution of rabbit anti-cobra antiserum (or) rabbit anti individual venom proteins antiserum for 1 h. This membrane was washed three times in PBS-containing 0.05% Tween-20 followed by washing with PBS 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 magnesium chloride (MgCl₂). Same molecular weight markers were used in the gel electrophoresis.

Purification of monospecific antisera

Hyperimmune sera raised against the venoms of Indian cobra (14, 29, 65, 72, and 99 kDa proteins) were purified by affinity chromatography using protein A sepharose™ CL-4B as described by Goding.²⁰ In brief, protein A sepharose (1.5 g) was swollen in borate-buffered saline (BBS), pH 8.0, for 1 h at room temperature, and the material when settled was transferred to a column of approximately 6–1.5 cm. The monospecific antiserum was dialyzed against BBS, and 2 ml of diluted antiserum was passed through the column. The column was washed thoroughly with BBS and bound IgG was then eluted with 4 M MgCl₂. The absorbance of each fraction was monitored at 280 nm. Protein-rich fractions were pooled, dialyzed against BBS, and the lyophilized powder was stored at −70.8°C.

Determination of LD₅₀

The lethal toxicity was determined in male Swiss strain mice. Groups of six animals were injected intraperitoneally (i.p.) with 0.5 ml of 0.85% NaCl containing increasing concentrations of cobra venom following the method described by Meier and Theakston²¹ Different concentrations of venom used for the determination of median lethal dose (LD₅₀) value for the route was 6.25, 7.81, 9.76, 12.20, 15.25, and 19.90 µg/animal. The dose that killed 50% of animals within 24 h after injection of venom was determined by Spearman–Karber method.²²

Intraperitoneal administration of antiserum at 0, 1, 2, 4, 6, 8, and 10 h after injection of N. 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 µg/animal) of N. n. naja venom and served as positive control. In group II, the mice were administered with N. n. naja venom and whole venom antiserum (400 µl/mouse, i.p.) simultaneously In groups III, IV, V, VI, VII, VIII, and IX, the mice were injected with N. n. naja venom (25 µg/mouse, i.p.). The mice in group III were administered with whole venom antiserum (400 µl/mouse, i.p.) 1 h after venom injection, while in groups IV, V, VI, VII, and VIII antiserum was administered after 2, 4, 6, 8, and 10 h, respectively. 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 triplicates. The volume of cocktail antiserum and commercial antiserum used in this experiment were 300 and 400 µl/mouse.

Mouse specimens

LD₅₀ of N. n. naja venom (12.37 mg/kg) was administered subcutaneously (s.c.) to mice. Three experimental mice and two normal controls were used at each time interval. Different organs (brain, heart, lungs, liver, spleen, and kidneys) as well as tissues at the site of injection were collected at various time intervals (0, 2, 4, 6, 8, 10, 12, and 24 h) after venom injection. The tissues were weighed and homogenized in PBS, pH 7.4 (100 mg/ml) using a homogenizer (Virtis, Warminster, Pennsylvania, USA) for 10 min at 10,000 r/min. The homogenized materials were centrifuged at 30,000g for 10 min, and the clear supernatant in each case was analyzed for the presence of venom by ELISA. Tissues from normal mice collected at similar time intervals and processed identically were used as control.

All animal experiments followed the instruction by Committee for the Purpose of Control and Supervision on Experiments on Animals, and the experimental mice were killed by cervical dislocation with well-trained lab technician.

ELISA for the detection of venom in envenomated mice

An experiment was conducted to make use of antisera raised against different proteins of cobra venom or whole venom antiserum to detect the venom in different organs of envenomated mice at different time intervals using ELISA. The organs such as heart, liver, kidney, brain, lungs, and spleen were dissected out from the envenomated mice at 0, 2, 4, 6, 8, 10, 12, and 24 h after injection of venom. Blood and exudates at the site of injection were also collected along with other samples. The organs were washed with PBS to eliminate blood contamination and then homogenized in PBS for 10 min. The ratio of tissue and buffer was 1: 5 (1 g in 5 ml of PBS). The homogenate was centrifuged at 10,000 r/min for 30 min at 4°C, and the clear supernatant was subjected to venom detection test. Tissue from normal mice injected with physiological saline served as negative controls. The wells of microtiter plate were coated at 5 µg/well with blood and tissue samples of envenomated mice collected at different time intervals after estimating protein concentration. The venom was detected by ELISA using anti-14 kDa protein of cobra venom. The optical density was measured at 405 nm using an automated ELISA reader, and the titers were determined.²³

Quantitation of venoms

In order to quantitate the amount of venom present in tissue samples, a titration curve was constructed by plotting the log of venom concentration against absorbance value for each snake venom. Known amounts of venom spiked with tissue supernatants from normal controls were included with each test to obtain a titration curve. Quantitation of each venom was carried out by working out the difference in the absorbance values between the experimental and control wells and by subsequent comparison with the titration curve. The venom concentration was calculated and expressed in terms of nanogram per milliliter of the tissue homogenate of mice.

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. The value of p < 0.05 was accepted as significant. Statistical calculations were performed using Statistical Package for Social Sciences (version 9, SPSS, Illinois, Chicago, USA) software.

Results

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

The concentration of protein in the venom of spectacled cobra was 62.56 ± 0.59 µg/µl. The protein profile of cobra venom was studied using SDS-PAGE. Figure 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 was calculated based on the standard protein markers such as 14, 24, 29, 45, 48, 65, 72, and 99 kDa, respectively (Figure 1).

Figure 1.

SDS-PAGE showing the protein pattern of Indian spectacled cobra. (Naja naja) venom. Lane M: marker; lanes 1 to 12: Naja naja venom. SDS: sodium dodecyl sulfate; PAGE: polyacrylamide gel electrophoresis.

Western blot analysis

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 concentrations of protein in the samples eluted from the SDS-PAGE corresponding to bands I, II, III, IV, V, VI, VII, and VIII were 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 µg/µl, respectively. The production of antisera was confirmed by Western blot analysis (Figure 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).

Figure 2.

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

Immunization and antibody response

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

Table 1.

Antibody titers of whole venom antiserum and individual venom proteins antisera of cobra venom.^a

Groups	Antibody dilution
I	5,12,000
II	No reaction
III	2,56,000
IV	16,000
V	No reaction
VI	1,28,000
VII	1,28,000
VIII	1,28,000
Whole venom	1,28,000

ELISA: enzume-linked immunosorbent assay; OD: optical density.

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

Determination of LD₅₀

The LD₅₀ value of cobra venom was determined in the mice model by different routes, and the results are shown in Table 2. For intraperitoneal route, the LD₅₀ was found to be 13.73 µg/mouse. The lower and upper limits were 12.03 and 15.55 µg/mouse, respectively.

Table 2.

LD₅₀ values of cobra venom in mice by different routes of injection at 24 h post injection.

Route of injection	LD₅₀ (µg/mouse)
Route of injection	Lower limit	Upper limit
Intraperitoneal	13.73
Intraperitoneal	12.03	15.55
Intravenous	8.44
Intravenous	7.35	9.53
Intramuscular	13.37
Intramuscular	12.03	15.55
Subcutaneous	13.73
Subcutaneous	12.06	15.55

LD₅₀: median lethal dose.

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

In total, 100% survival was observed in envenomated mice treated with whole venom antiserum at the dose of 400 µl/mouse at 0, 1, 2, and 4 h after injection of cobra venom, and 83.32% survival was observed in mice administered with 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 µl/mouse, and 100% survival was observed in the case of mice treated with cocktail antiserum (300 µl/mouse) at 0, 1, 2, 4, and 6 h after injection of venom (Table 3).

Table 3.

Percentage survival of envenomated mice treated with different types of antisera at different time intervals (in hours) after injection of cobra venom. (n = 18).

Antiserum type	Concentrationof venom (µg/mouse) used	Percentage of survival at 24 h after administration of the respective antiserum
Antiserum type	Concentrationof venom (µg/mouse) used	0 (h)^a	1 (h)^a	2 (h)^a	4 (h)^a	6 (h)^a	8 (h)^a	10 (h)^a
Whole venom antiserum (400 µl/animal)	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
Cocktail antiserum (300 µl/animal)	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
Commercial antiserum (400 µl/animal)	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
Positive control (PBS and venom)	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
Negative control (PBS only)	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

PBS: phosphate-buffered saline.

^aAdministration of antiserum at 0, 1, 2, 4, 6, 8 and 10 h after injection of cobra venom. The values are represented as means ± SE.

ELISA for the detection of N. n. naja venom in envenomated animals

The ELISA technique was used to detect the venom in envenomated mice. The organs (heart, liver, kidney, brain, lungs, and spleen) of envenomated mice were used for the screening of venom. The organs were collected at different time intervals (1, 2, 4, 6, 8 10, 12, and 24 h or following death stage after post injection of venom) and used to detect the venom using ELISA. Blood and exudates at the site of injection were also collected along with other samples for screening purpose. The results are presented in Table 4. The venom was detected in all the organs of envenomated mice at 4 h post injection of venom. The venom was detected in the blood of envenomated mice only up to 6 h post injection, and it showed negative at 8 h of post injection. The venom was detected in all other organs up to following death stage.

Table. 4.

Detection of Indian cobra venom in different tissues obtained from the experimental envenomated mice injected with lethal dose of venom by ELISA at different time intervals.

Groups (n = 6)	Venom injected	Number of positive/tested
Groups (n = 6)	Venom injected	Blood	Brain	Heart	Liver	Kidney	Lungs	Spleen	Site of Injection
I (1 h)	Naja naja	6/6	6/6	6/6	6/6	6/6	6/6	6/6	6/6
II (2 h)	N. naja	6/6	6/6	6/6	6/6	6/6	6/6	6/6	6/6
III (4 h)	N. naja	5/6	6/6	6/6	6/6	6/6	6/6	6/6	5/6
IV (6 h)	N. naja	5/6	5/6	6/6	6/6	6/6	6/6	6/6	6/6
V (8 h)	N. naja	0/6	5/6	6/6	6/6	6/6	5/6	6/6	5/6
VI (moribund stage)	N. naja	0/6	4/6	6/6	6/6	6/6	5/6	5/6	5/6
Negative control	PBS	0/6	0/6	0/6	0/6	0/6	0/6	0/6	0/6

PBS: phosphate-buffered saline; ELISA: enzyme-linked immunosorbent assay.

Quantitation of N. n. naja venom in mice

The titration curve for the quantitation of known concentrations of N. n. naja venom showed that the assay can detect venom levels up to 2.5 ng/ml of tissue homogenate. The venom concentrations in various organs of mice as well as tissue at the site of injection at various time intervals following death are shown in Figure 3. Tissue at the site of injection showed the highest concentration of venom, whereas brain showed the least. Heart, liver, kidneys, spleen, and lungs showed moderate concentrations in the increasing order. A correlation was found between the amount of venom detected and time elapsed. A large quantity of venom was detected at 24 h after injection in all tissues. The venom levels decreased gradually up to 24 h at the site of injection and blood. Tissue at the site of injection showed a high level of venom even following death stage, suggesting that the tissue at the site of envenomation may serve as a best source for venom detection in human cadavers too.

Figure 3.

Naja naja venom levels in different tissues of mice at various time intervals following death. Mice were intraperitoneally injected with 200 μl of PBS at pH 7.4 containing LD₅₀ of venom; the supernatants of tissue samples were analyzed in duplicates. Venom concentrations (in nanogram per milliliter) were interpolated from a titration curve of N. naja venom diluted in tissue homogenates from control mice. Results are presented as mean ± SD (n = 6). PBS: phosphate-buffered saline; LD₅₀: median lethal dose.

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, 24, 29, 45, 48, 65, 72, and 99 kDa were observed on SDS-PAGE in the venom of N. n. naja. Similar type of protein banding pattern was observed by many workers.^24

–27 Mendoza et al. observed 7 protein bands in SDS-PAGE and 12 bands in non-SDS electrophoresis.²⁴ SDS-PAGE analysis was carried out on Indian cobra venom obtained from 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, 20, 24, 55, 105, and 110 kDa.²⁶ 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 with monovalent antivenoms.^28,29 Hence, an alternate technology should be explored to produce suitable antivenom. In the present study, an attempt was made to make use of a cocktail antiserum prepared by mixing 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 CFA was used.³⁰ 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. Chotwiwatthanakun et al. 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.³¹

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.^32

–36 All these workers have tried to generate polyclonal antibodies in chicken egg against venoms of cobra, krait, and vipers. Almeida et al. 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.³² Anti-viper venom antibodies were raised in Rhode Island Red inbred hens, and these antibodies were isolated using a simple method. These antibodies showed good antigen binding and neutralization of venom in in vitro assays.³³ Thalley and Carroll have generated antivenoms against rattlesnake and scorpion venoms in chicken egg yolk and their neutralizing activity was demonstrated by in vivo experiments using mice.³⁴ Antivenom against saw-scaled viper was generated in the egg of white leghorn chickens as an alternative to conventional polyvalent equine antivenom.³⁵ Meenatchisundaram et al. have used chickens as an alternative source of antibody production against cobra, krait, Russell’s viper, and saw-scaled viper venoms.³⁶

The antiserum raised against venom protein of 14 kDa was used to detect the venom in different organs of envenomated mice by ELISA. The results showed that the antiserum could detect the venom in most of the organs tested at moribund stage of experimental animals. The ELISA test kit developed by Dong et al. was used to detect the venom of four medically important snakes in Vietnam.³⁷ In the present study, the ELISA technique was used to detect the venom in different organs of envenomated mice using the antiserum raised against single protein of cobra venom. The venom in different organs was determined qualitatively not quantitatively. Selvanayagam et al. developed a double antibody sandwich ELISA to detect the viper, E. carinatus venom in various organs of envenomated mice at different time intervals_. ³⁸

For the detection of snake venoms, there are several reports on clinical cases of envenomed victims but only a few studies were carried out on autopsy specimens. In the present study, we developed a sandwich ELISA to measure N. n. naja venom in various tissue samples of mice. The venom was detected in all the organs tested, indicating the distribution of N. n. naja venom in all vital organs and not in any specific organ (Figure 3). However, concentrations of venom varied from one organ to another. The highest concentration observed at the site of injection suggested that the venom slowly entered into blood circulation from the site of injection. Moderate amounts of venom in kidneys indicated possible renal excretion of venom; similar results were obtained with D. r. russelli ³⁹ and Vipera ammodytes venoms.⁴⁰ Low concentrations in the brain showed that the blood–brain barrier has some role in preventing the movement of venom into the brain. This may also prove logical in view of the fact that N. n. naja venom contains toxic proteins that mainly affect the hemostatic mechanism rather than the central nervous system.

Several sensitive ELISAs have been reported for the detection of snake venoms in human victims.^41

–47 The increase in sensitivity may be attributed to the avidin/biotin amplification, which enables the detection of extremely low levels of venom antigens in autopsy specimens. In venom detection, nonspecific reactivity was observed,^47,48 and the severity of the problem was elaborately discussed by Ho et al.⁴¹ In the present investigation, this problem of nonspecific binding was not encountered when normal mice autopsy samples were used. The AB-microELISA reported here is not designed as an emergency investigation in clinical medicine, since the complete procedure requires a minimum of 2 h and 10 min after blocking.⁴⁹ However, it is proposed to develop a simple, rapid, and species-specific immunodiagnostic kit.

Conclusion

In conclusion, these data suggest that the present investigation have thrown up the possibility of developing alternate technologies for the production of antivenom against cobra venom. These results demonstrated that the optimized ELISA was adequate to quantify cobra venom levels in different biological samples. 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.

Footnotes

Acknowledgment

The authors thank the management of C. Abdul Hakeem College, Melvisharam, Vellore District, Tamil Nadu, India, for providing facilities to carry out this work. The authors also thank the Secretary, Irula Snake Catcher’s Cooperative Society, Chennai, Tamil Nadu, India, for providing the snake venoms for this study and King’s Institute of Preventive Medicine for providing snake venom antiserum. The authors are grateful to Mr P. Thomas John, Former Head, Department of Zoology, C. Abdul Hakeem College, Melvisharam, Vellore District, Tamil Nadu, India.

Funding

The author CV is a recipient of Young Scientist award from the Department of Science and Technology, New Delhi, India.

References

Ananthapadmanabhan

Snake bite. In: Dalal

(ed) Medicine update. Bombay, India: Association of Physicians of India, 1991, pp. 93–114.

Whitaker

. Common Indian snakes: a field guide. Madras, India: MacMillan India, 1978.

Deoras

. Snakes of India. New Delhi, India: National Book Trust, 1990.

Jacob

. Diagnosis and management of snake venom poisoning. Bombay, India: Varghese Publishing House, 1990.

Jadhav

Kapre

SV.

Antivenin production in India. In: Tu

(ed) Reptile venoms and toxins: Handbook of natural toxins, Vol. 5. New York, NY: Marcel Dekker, 1991, pp. 583–610.

Thachil

Tony

Jude

Ross

Vincent

NDJ

Sridhar

. Antisnake venom: an over-used medication. Trop Doctor 1992; 22(3): 113–115.

Jena

Sarangi

. Snakes of medical importance and snake bite treatment. New Delhi, India: Ashish Publishing House, 1993.

Theakston

. The application of immunoassay techniques, including enzyme-linked immunosorbent assay (ELISA), to snake venom research. Toxicon 1983; 21(3): 341–352.

Warrell

Bidwell

Voller

. A critical reappraisal of the use of enzyme-linked immunosorbent assays in the study of snake bite. Toxicon 1986; 24(3): 211–221.

10.

Minton

. Present tests for detection of snake venom: clinical applications. Ann Emerg Med 1987; 16(9): 932–937.

11.

Ratanabanangkoon

Billings

Matangkasombut

. Immunodiagnosis of snake venom poisoning. Asian Pacific J Aller Immunol 1987; 5: 187–190.

12.

Laemmeli

. Cleavage of structural proteins during the assembly of the head of bacteriophage T₄ . Nature 1970; 227: 680–685.

13.

Lowery

Rosebrough

Farr

Randall

. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–275.

14.

Hunkapiller

Lujan

Purification of microgram quantities of proteins by polyacrylamide gel electrophoresis. In: Shively

(ed.) Methods of protein microcharacterization. Clifton, NJ: Humana Press, 1986, pp. 89–101.

15.

Nadala

ECB

Jr Tapay

Cao

Loh

. Detection of yellowhead virus and Chinese baculovirus in penaeid shrimp by the Western blot technique. J Virol Methods 1997; 69 (1–2): 39–44.

16.

Ramana

Capoor

Sashidhar

Bhat

. Limitations in the use of horseradish peroxidase as an enzyme probe in the development of homogeneous immunoassay for aflatoxin B1. J Anal Chem 1995; 352 (1–2): 43–48.

17.

Sambrook

Fritsch

Maniatis

. Molecular cloning: a laboratory manual. 2nd ed. NY: Cold Spring Harbor Laboratory, Cold Spring Harbor, 1989.

18.

Zang

. The use of gene specific IgY antibodies for drug target discovery. Drug Discov Today 2003; 8(8): 364–371.

19.

Towbin

Staehelin

Gordon

. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications. Proc Natl Acad Sci USA 1979; 76: 4350–4354.

20.

Goding

. Conjugation of antibodies with fluorochromes: modifications to standard methods. J Immunol Methods 1976; 13 (3–4): 215–226.

21.

Meier

Theakston

. Approximate LD₅₀ determinations of snake venoms using eight to ten experimental animals. Toxicon 1986; 24(4): 395–401.

22.

World Health Organization. Progress in the characterization of venoms and standardization of antivenoms. WHO Offset Publication no. 58 . Geneva, Switzerland: World Health Organization, 1981, 1–44.

23.

Schurrs

AHWM

Van Weeman

. Enzyme-immunoassay. Clin Chim Acta 1977; 81: 1–40.

24.

Mendoza

CEC

Bhatti

. Electrophoretic analysis of snake venoms. J Chromatogr B Biomed Sci Appl 1992; 580 (1–2): 355–363.

25.

Sashidharamurthy

Jagadeesha

Girish

Kemparaju

. Variations in biochemical and pharmacological properties of Indian cobra (Naja naja naja) venom due to geographical distribution. Mol Cell Biochem 2002; 229 (1–2): 93–101.

26.

Brunda

Sashidhar

Sarin

. Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin. Toxicon 2006; 48(2): 183–194.

27.

Chellapandi

Jebakumar

SRD

. Purification and antibacterial activity of indian cobra and viper venoms. Electr J Bio 2008; 4(1): 11–16.

28.

Premawardhena

De-Selva

Fonseka

MMD

Gunatilake

de-Silva

. Low dose subcutaneous adrenaline to prevent acute adverse reactions to antivenom serum in people bitten by snakes; randomized, placebo controlled trial. Br Med J 1999; 318: 1041–1043.

29.

Theakston

RDG

Warrell

Griffiths

. Report of a WHO workshop on the standardisation and control of antivenoms. Toxicon 2003; 41: 541–547.

30.

Pratanaphon

Akesowan

Khow

Sriprapal

Pratanaphon

. Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia). Vaccine 1997; 15(14): 1523–1528.

31.

Chotwiwatthanakun

Pratanaphon

Akesowan

Sriprapat

Tatanabanangkoon

. Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol. Toxicon 2001; 39(10): 1487–1494.

32.

Almeida

CMC

Kanashiro

Rangel Filho

Mata

MFR

Kipnis

Dias da Silva

. Development of snake antivenom antibodies in chickens and their purification from yolk. Vet Rec 1998; 143: 579–584.

33.

Devi

Bai

Lal

Umsahanker

Krishana

. An improved method for isolatin of antiviper venom antibodies from chicken egg yolk. J Biochem Biophys Methods 2002; 51(2): 129–138.

34.

Thalley

Carroll

. Rattlesnake and scorpion antivenin from the egg yolks of immunized hens. Nature Biotechnol 1990; 8: 934–938.

35.

Paul

Manjula

Deepa

Selvanayagam

Ganesh

Subba Rao

. Anti Echis carinatus venom antibodies from chicken egg yolk isolation, purification and neutralization efficacy. Toxcion 2007; 50(7): 893–900.

36.

Meenatchisundaram

Parameswari

Michael

Ramalingam

. Neutralization of the pharmacological effects of Cobra and Krait venoms by chicken egg yolk antibodies. Toxicon 2008; 52(2): 221–227.

37.

Dong

Quyen

Eng

Gopalakrishnakone

. Immunogenicity of venoms from four common snakes in the South of Vietnam and development of ELISA kit for venom detection. J Immunol Methods 2003; 282 (1–2): 13–31.

38.

Selvanayagama

Gnanavendhana

Ganesh

Rajagopal

Rao

. ELISA for the detection of venoms from four medically important snakes of India. Toxicon 1999; 37(5): 757–770.

39.

Than

Thwin

San

. Distribution of ¹²⁵I-labelled Russell’s viper (Vipera russelli) venom in mice. Snake 1985; 17: 124–130.

40.

Labrousse

Nishikawa

Bon

Avrameas

. Development of a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite. Toxicon 1988; 26(12): 1157–1167.

41.

Warrell

Looareesuwan

Phillips

Chanthavanich

Karbwang

. Clinical significance of venom antigen levels in patients envenomed by the Malayan pit viper (Calloselasma rhodostoma). Am J Trop Med Hyg 1986; 35(3): 579–587.

42.

Audebert

Grosselet

Sabouraud

Bon

. Quantitation of venom antigens from European vipers in human serum or urine by ELISA. J Anal Toxicol 1993; 17(4): 236–240.

43.

Audebert

Sorkine

Bon

. Envenoming by viper bites in France: clinical gradation and biological quantification by ELISA. Toxicon 1992; 30 (5–6): 599–609.

44.

Bucher

Canonge

Thomas

Tyburn

Vincent

Choumet

. Clinical indicators of envenoming and serum levels of venom antigens in patients bitten by Bothrops lanceolatus in Martinique. Trans R Soc Trop Med Hyg 1997; 91: 186–190.

45.

Chavez-Olortegui

Lopes

Cordeiro

Granier

Diniz

. An enzyme-linked immunosorbent assay (ELISA) that discriminate between Bothrops atrox and Lachesis muta muta venoms. Toxicon 1993; 31(4): 417–426.

46.

Chavez-Olortegui

Penaforte

Silva

Ferreira

Rezende

Amaral

CFS

. An enzyme-linked immunosorbent assay (ELISA) that discriminates between the venoms of Brazilian Bothrops species and Crotalus durissus . Toxicon 1997; 35(2): 253–260.

47.

Sjostrom

Karlson-Stiber

Persson

Al-Abdulla

Smith

. Development and clinical application of immunoassays for European adder (Vipera berus berus) venom and antivenom. Toxicon 1996; 34(1): 91–98.

48.

Coulter

Harris

Sutherland

. Enzyme immunoassay for the rapid clinical identification of snake venom. Med J Aust 1980; 1(9): 433–435.

49.

Dhaliwal

Lim

Sukumaran

. A double antibody sandwich micro-ELISA kit for the rapid diagnosis of snake bite. Southeast Asian J Trop Med Public Health 1983; 14(3): 367–373.

Detection and neutralization of cobra venom using rabbit antiserum in experimental envenomated mice

Abstract

Keywords

Introduction

Materials and methods

Venom and experimental animals

Chemicals

SDS-PAGE analysis of snake venom

Electroelution of different proteins of N. n. naja venom

Production of antiserum

ELISA for the determination of anti-snake venom antibodies

Western blot analysis

Purification of monospecific antisera

Determination of LD50

Intraperitoneal administration of antiserum at 0, 1, 2, 4, 6, 8, and 10 h after injection of N. n. naja venom

Mouse specimens

ELISA for the detection of venom in envenomated mice

Quantitation of venoms

Statistical analysis

Results

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

Western blot analysis

Immunization and antibody response

Determination of LD50

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

ELISA for the detection of N. n. naja venom in envenomated animals

Quantitation of N. n. naja venom in mice

Discussion

Conclusion

Footnotes

Acknowledgment

Funding

References

Determination of LD₅₀

Determination of LD₅₀