International Immunopharmacology 21 (2014) 148–155

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International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp

Cross-reactivity and neutralization of Indian King cobra (Ophiophagus

hannah) venom by polyvalent and monovalent antivenoms
Yashonandana J. Gowtham a, Y.H. Mahadeswaraswamy a, K.S. Girish b, Kemparaju K. a,⁎
a
Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysore 570006, Karnataka, India
b
Department of Studies in Biochemistry, Tumkur University, Tumkur, Karnataka, India

a r t i c l e i n f o a b s t r a c t

Article history: The venom of the largest venomous snake, the king cobra (Ophiophagus hannah), is still out of league for the pro-
Received 25 September 2013 duction of therapeutic polyvalent antivenom nor it is characterized immunologically in the Indian subcontinent.
Received in revised form 20 March 2014 In the present study, the king cobra venom is comparatively studied for the cross-reactivity/reactivity and toxic-
Accepted 10 April 2014
ity neutralization by the locally available equine therapeutic polyvalent BSV and VB antivenoms, and monovalent
Available online 9 May 2014
antivenom (OH-IgG) prepared in rabbit. None of the two therapeutic antivenoms procured from two different
Keywords:
firms showed any signs of cross-reactivity in terms of antigen–antibody precipitin lines in immunodouble diffu-
Snake venom sion assay; however, a weak and an insignificant cross-reactivity pattern was observed in ELISA and Western blot
Ophiophagus hannah venom studies. Further, both BSV and VB antivenoms failed to neutralize proteolytic, hyaluronidase and phospholipase
Immunological cross-reactivity activities as well as toxic properties such as edema, myotoxicity and lethality of the venom. As expected, OH-IgG
Venom neutralization showed strong reactivity in immunodouble diffusion, ELISA and in Western blot analysis and also neutralized
Antivenom both enzyme activities as well as the toxic properties of the venom. Thus, the study provides insight into the likely
measures that are to be taken in cases of accidental king cobra bites for which the Indian subcontinent is still not
prepared for.
© 2014 Elsevier B.V. All rights reserved.

1. Introduction
Snakebite is one of the major health hazards that prevail in many
parts of the world, especially in the tropical developing countries [1].
The total number of annual snakebites across the world has been esti-
mated to be 5.4 million, with approximately 100,000 deaths in Asia
alone [2]. In India, there are more than 200,000 snakebite cases, of
which approximately 35,000 to 50,000 people die annually [3]. As of
now, antivenom therapy is the common mode of treatment in the sub-
continent. The antivenom is of polyvalent nature and made in horses for
the venoms of the endemic snakes such as cobra (Naja naja), Russell's
viper (Daboia/Vipera russellii), saw-scaled viper (Echis carinatus) and
krait (Bungarus caereleus). The therapy is expected to offer protection
against bites by all big four snakes. This is because in most cases the bit-
ten species remained mysterious. However, in recent years two more
snake species, the king cobra (Ophiophagus hannah) and the hump-
nosed pit viper (Hypnale hypnale) are drawing attention and have also
been considered as important poisonous snakes in addition to the big
four of the subcontinent.
The king cobra usually inhabits the dense forest zones of Western
ghats as well as the hilly regions in the eastern parts of India and thus
⁎ Corresponding author at: Department of Studies in Biochemistry, University of
Mysore, Manasagangothri, Mysore 570006, Karnataka, India. Tel.: +91 821 2419625.
E-mail address: kemparajuom@gmail.com (K. K.).
normally keeps away from human habitat. Hence, threat to human life
is generally not warranted. More so, being the biggest poisonous
snake it is believed to inject a large quantity of venom that will ensure
easy killing of the victim and hence any attempt made to treat the ven-
omous bite appears futile.
It appears that these reasons restricted king cobra venom from being
studied for its biochemical, pharmacological, immunological and
toxicity neutralization properties. Although few toxins [4–12] have
successfully been isolated and characterized from the venom, there
still remained an incredible scope for exploring the venom for the said
properties. Such studies not only establish and quantify the various
properties of the venom, but also provide vital information for the pro-
duction of life saving therapeutic antivenom. Because of the rapid
growth and developmental activities involving deforestation and en-
croachments, the thick forest zones are vanishing and thus the habitat
of king cobra as well. Therefore, it is highly likely that there will be
king cobra and human encounters like any other poisonous snakes of
the subcontinent in the years to come. Thus, it becomes essential to ex-
plore the king cobra venom neutralizing efficacy of the commercial
therapeutic polyvalent antivenoms available. This not only validates
the neutralizing efficacy of therapeutic polyvalent antivenom, but also
offers scope for prior preparedness in terms of the production of king
cobra antivenom so that attempts can be made to save the accidental
king cobra bite victims if the situation demands and hence the present
study.

http://dx.doi.org/10.1016/j.intimp.2014.04.012
1567-5769/© 2014 Elsevier B.V. All rights reserved.
 Y.J. Gowtham et al. / International Immunopharmacology 21 (2014) 148–155
2. Materials and methods
O. hannah venom was obtained from the sole distributor Mr. Dipak
Kumar Mitra, Hindustan Park, Kolkata, India. Venoms of D. russellii,
E. carinatus and N. naja were obtained from Irula Snake Catchers Co-
operative Society, Kanchipuram district, Tamilnadu, India. Commercial
snake venom antisera from Bharath serum and vaccines (BSV),
Ambernath, India, and Vins Bioproducts (VB), Andhra Pradesh, India
were obtained from local drug dealers. Anti-equine goat IgG secondary
antibody and molecular weight markers were obtained from Genei Bio
products, Bangalore, goat anti-rabbit IgG, fat free casein, agarose gel,
immobilon-PVDF membranes, luminol, p-coumaric acid, Freund's com-
plete and incomplete adjuvants, and hyaluronic acid were obtained
from Sigma Chemicals Pvt. Ltd., St Louis, USA. Protein-A Sepharose CL-
6B was obtained from Pharmacia, Sweden. Certified New Zealand
white albino rabbit was obtained from licensed breeders and Swiss
Wistar albino mice were obtained from Central Animal House, Universi-
ty of Mysore by the clearance from the Animal Ethics Committee,
CPCSEA, Chennai and permission from Central Animal House, Dept. of
Zoology, University of Mysore, Mysore. Animal care and handling
were conducted in compliance with the national regulations for animal
research. Fresh blood samples were collected from healthy human vol-
unteers with the permission and clearance from the Human Ethics Com-
mittee. All other chemicals used otherwise were of analytical grade.
2.1. Immunization of rabbit and purification of OH-lgG
O. hannah venom (100 μg) in 0.2 ml phosphate buffered saline (PBS)
was mixed with equal volumes of Freund's adjuvants and used for immu-
nization of rabbits as described by Shashidharamurthy et al. (2002). Blood
was drawn from the marginal ear vein of rabbits. Part of the serum (OH-
antiserum) was set apart for titer value determination. About 10 ml of the
OH-antiserum was subjected to ammonium sulphate fractionation and
processed to obtain crude immunoglobulin G fraction as described by
Heide and Schwick [13]. It was then subjected to Sepharose CL-6B affinity
column chromatography. The column with 2 ml bed volume was equili-
brated with PBS and 10 mg crude immunoglobulin G fraction in 3 ml
PBS was loaded. The column was washed with PBS and then eluted
using 0.2 N glycine–HCl buffer pH 2.9. Aliquots of 1 ml were collected
and pooled after reading optical density at 280 nm. It was then neutral-
ized using 1 N Tris–HCl buffer pH 8.0 and it was subjected to SDS-PAGE
under both reduced and non-reduced conditions.
2.2. Cross-reactivity studies
2.2.1. Ouchterlony immunodouble diffusion assay
The technique of Williams and Chase [14] was followed to know the
cross reactivity. Diffusion plates were prepared by pouring 7 ml of 0.75%
molten agarose in saline into Petri dishes. After solidification, gels were
punched with the help of a punching tool to make even sized central
and peripheral wells. The central well was filled with 400 μg each of
OH-IgG/polyvalent antivenoms (BSV/VB), while peripheral wells were
filled with 200 μg each of four different snake venoms. The plates
were kept at 37 °C for about 24 h and then photographed.
2.2.2. ELISA
ELISA was performed using 96 well plates. O. hannah venom
1 μg/100 μl carbonate-bicarbonate buffer, 0.2 M, pH 9.6 was coated
to the wells and incubated overnight at 4 °C. Blocking buffer (car-
bonate-bicarbonate buffer, 0.2 M, pH 9.6 containing 5% skimmed
milk and 1% bovine serum albumin) was added to wells and incu-
bated for 2 h at 37 °C. Wells were washed with PBS containing
0.2% Tween-20 (wash buffer). Then, serially diluted BSV,VB and
OH-IgG were independently added and incubated at 37 °C for
45 min (BSV, VB and OH-IgG, 180 μg each in 100 μl PBS were serially
diluted independently in PBS and used) followed by washing with
149
wash buffer. Secondary antibodies (anti horse goat IgG/anti-rabbit
goat IgG), conjugated with horse radish peroxidase (1:10,000 dilu-
tion) was added and incubated for 45 min at 37 °C. Undiluted chro-
mogenic substrate (100 μl) 3,3′,5,5′-tetra methyl benzidine (TMB)
was added and incubated at room temperature for 30 min. The reac-
tion was stopped by adding 100 μl of 1 N H2SO4. The color was read
at 405 nm, using a Biotek ELx 800-ELISA plate reader.
2.2.3. Western blot
SDS-PAGE (10%) was carried out according to the method of
Laemmli [15]. using Tris (25 mM), glycine (192 mM) and SDS (0.1%)
for 3 h at 90 V at room temperature. After electrophoresis, the gel was
removed and placed on a PVDF membrane and was made into a sand-
wich by placing blotting papers on both the sides. The sandwich was
placed inside the blot transfer unit containing Tris–glycine transfer buff-
er 0.12 M, pH 8.3. Blotting onto the membrane was carried out for
45 min at a constant current of 50 V. The membrane was taken out,
washed with Tris buffer 0.12 M, pH 8.0 and blocked with a blocking
buffer (carbonate–bicarbonate buffer, 0.2 M, pH 9.6 containing 5%
skimmed milk and 1% bovine serum albumin). Polyvalent antivenom
(BSV/VB) and monovalent OH-IgG were added independently in suit-
able dilution using Tris buffer 0.12 M, pH 8.0 onto the membrane blot
followed by the addition of secondary antibody (anti equine goat anti-
body conjugated with horse-radish peroxidase/anti-rabbit goat IgG
with horse-radish peroxidase). The blot was developed on a photo-
graphic film in a dark room using an enhanced chemi-luminescence
kit containing luminol and p-coumaric acid. The film was washed,
dried and photographed.
3. In vitro neutralization studies
3.1. Proteolytic activity
The proteolytic activity was assayed according to the method of
Satake et al. [16]. O. hannah venom (200 μg) was pre-incubated with
varying amounts of antivenoms BSV/VB/OH-IgG for 30 min at 37 °C.
Fat free casein (0.4 ml, 2% in 0.2 M Tris–HCl buffer, pH 8.5) was incubat-
ed with pre-incubated samples in a final volume of 1 ml at 37 °C for 2 h
30 min. The reaction was stopped by adding 1 ml 0.44 M Trichloroacetic
acid and was left to stand for 30 min. The mixture was then centrifuged
at 1500 g for 10 min. Sodium carbonate (2.5 ml, 0.4 M) and Folin–
Ciocalteau's reagent (1:2 dilution with water) was added to 1 ml of
the supernatant and the color developed was read at 660 nm. One
unit of enzyme activity was defined as the amount of enzyme required
to cause an increase in OD of 0.01 at 660 nm/min at 37 °C. The specific
activity was expressed as units/min/mg protein.
3.2. Hyaluronidase activity
The hyaluronidase activity was assayed according to the method of
Reissig et al. [17] using pre-incubated samples that consisted of
O. hannah venom (200 μg) with varying amounts of antivenoms BSV/
VB/OH-IgG. The pre-incubated samples were incubated with hyaluronic
acid 100 μg in 300 μl 0.2 M sodium acetate buffer, pH 5.5 containing 0.15
M NaCl for 2 h 30 min at 37 °C. The reaction was terminated by adding
50 μl potassium tetraborate 0.1 M and boiled for 3 min. The coloring re-
agent p-dimethyl aminobenzaldehyde (PDMAB) 1.5 ml (acetic acid: hy-
drochloric acid; 9:1; v/v) was added and incubated for 30 min at 37 °C.
The absorbance was measured at 585 nm. Activity was expressed as
moles of N-acetyl glucosamine released/min/mg of protein.
3.3. Indirect hemolytic activity
Indirect hemolytic activity was determined according to the method
of Boman and Kaletta [18] using washed human erythrocytes. Briefly,
O. hannah venom (50 μg) was pre-incubated with varying amounts of
150 Y.J. Gowtham et al. / International Immunopharmacology 21 (2014) 148–155
BSV/VB/OH-IgG (50 μg to 500 μg) for 30 min at 37 °C. Packed human
erythrocytes, egg yolk and PBS (10 mM, pH 7.2 with 0.9% NaCl) were
mixed in the ratio of 1:1:8 (v/v); 1 ml of this suspension was incubated
with pre-incubated samples for 1 h at 37 °C. The reaction was stopped
by adding 9 ml of ice cold PBS and centrifuged at 1000 g for 10 min at
4 °C. The amount of hemoglobin released in the supernatant was mea-
sured at 540 nm. Activity was expressed as percent of hemolysis against
100% lysis of cells due to addition of water taken as positive control of
the experiment.
3.4. Plasma re-calcification time
The method of Quick et al. [19] was followed. The O. hannah venom
sample (40 μg) was pre-incubated with varying amounts (40 μg to
280 μg) of BSV/VB/OH-IgG for 30 min at 37 °C. Normal human citrated
plasma (0.2 ml) was then incubated with pre-incubated samples
taken in glass tubes and were mixed with Tris–HCl buffer 10 mM,
pH 7.5 to make up the final volume to 250 μl and was incubated at
37 °C for 1 min. The clotting time was determined after adding 0.1 ml
of 0.25 M CaCl2. The clotting time in the absence of venom samples
formed the control.
3.5. Myotoxicity
Myotoxicity was determined according to the method of Gutierrez
et al. [20] using groups of mice (n = 3). Group I was injected with lethal
dose25 (LD25) of O. hannah venom alone. Group II was injected with
pre-incubated venom–polyvalent antivenom (BSV), group III was
injected with pre-incubated venom–polyvalent antivenom (VB) and
Group IV was injected with pre-incubated venom–OH-IgG with increas-
ing ratios. Groups V and VI were injected with antibodies and saline re-
spectively. Mice were anesthetized by sodium barbiturate inhalation
after 3 h and the abdominal cavities were opened to draw the blood
from vena cava. After clotting, the 1:25 diluted serum was assayed for
creatine phosphokinase (CPK, EC no: 2.7.3.2) and lactate dehydrogenase
(LDH, EC no: 1.1.1.28) levels using AGAPEE diagnostic kits. Activities
were expressed as IU/L.
3.6. Edema
Venom sample (20 μg) was pre-incubated with varying amounts of
BSV/VB/OH-IgG for 30 min at 37 °C and injected into the right foot
pads of groups of mice (n = 3) separately. The left foot pads were
injected with the corresponding volumes of saline that served as con-
trols. The mice were sacrificed after 45 min and both the foot pads
were cut off at the ankle joints and weighed individually. Edema ratio
was calculated according to Vishwanath et al. [21]. The increase in
weight of leg due to edema was calculated as the edema ratio, which
equals the weight of edematous leg × 100/weight of normal leg. Mini-
mum edema dose (MED) was defined as the amount of protein required
to cause an edema ratio of 120%.
3.7. Lethality
LD50 of venom was determined according to the method of Meier
and Theakston [22] using (8–10) mice where the survival time was re-
corded for 24 h. For neutralization studies, twice the LD50 dose of
O. hannah venom/kg body weight was pre-incubated with increasing
ratio of BSV/VB/OH-IgG for 30 min at 37 °C. The samples were injected
(i.p) into groups of mice (n = 6). Groups of mice received antivenoms
alone served as controls. Documentation of survival time was done on
constant observation. Percent (%) survival was calculated as number
of mice died × 100 / number of mice used for the experiment (n).
3.8. Statistical analysis and IC50 value determination
Statistical analysis was done using Student's t-test to compare signif-
icant differences among the three antivenoms studied. P b 0.01 values
were considered as significant. All the data were presented as
mean ± SEM. The IC50 value was calculated considering the enzyme
activity of the venom sample in the absence of antibodies as 100%
and the amount of antibody that was required to inhibit 50% of the
activity as IC50 values.
4. Results
4.1. Cross-reactivity of polyvalent and monovalent antivenoms with four
snake venoms
The monovalent OH-antiserum prepared in rabbit for O. hannah
venom showed the titer value of 16,500 in ELISA. Ammonium sulfate
fractionation of the antiserum resulted in the crude immunoglobulin
G (IgG) fraction. The pure IgG was prepared by subjecting the crude
IgG fraction to affinity chromatography on a protein A Sepharose col-
umn and the purified IgG was designated as OH-IgG. The purity of
OH-IgG was confirmed by SDS-PAGE where a single band of about 150
kD molecular mass in non-reduced condition while two bands of low
molecular mass under reduced condition were revealed. In addition,
OH-IgG, and commercial therapeutic polyvalent antivenoms, BSV and
VB were compared for cross-reactivity and neutralization of common
snake venoms. BSV and VB antivenoms did not show any precipitin
bands with O. hannah venom (Fig. 1A well 1 and Fig. 1B well 1) while
they showed strong antigen–antibody precipitin bands with N. naja,
V. russellii and E. carinatus venoms (Fig. 1A and B). As expected, OH-
IgG showed strong conspicuous precipitin bands with O. hannah
venom (Fig. 1C well 1). Interestingly, OH-IgG did not show any precip-
itin bands with V. russellii and E. carinatus venoms (Fig. 1C) but, showed
detectable antigen–antibody precipitin bands with N. naja venom
(Fig. 1C well 2). Further, the extent of reactivity/cross-reactivity of
BSV, VB and OH-IgG antivenoms was determined by ELISA (Fig. 2).
Both BSV and VB showed strong reactivity with N. naja, E. carinatus
and V. russellii venoms but they showed feeble cross-reactivity with
O. hannah venom. The minimum amount of antivenoms required to de-
tect 1 μg each of N. naja, E. carinatus, and V. russellii venoms was found to
be in the range between 13 ± 0.55 ng and 22 ± 0.48 ng (Fig. 2A and B),
while for O. hannah venom it was found to be about 120 ± 0.66 ng
which accounts to about 10 folds less sensitivity as compared to the sen-
sitivity seen for the other three venoms. However, OH-IgG did not show
any sign of cross-reactivity with V. russellii venom but, it showed little
cross-reactivity with N. naja and E. carinatus venoms (Fig. 2C) with the
respective sensitivity of about 4 and 17 folds lesser than of O. hannah
venom. Fig. 3A shows the protein banding pattern of venoms in SDS-
PAGE, while Fig. 3B, C and D shows the reactivity/cross-reactivity pat-
tern of venom proteins with BSV, VB and OH-IgG antivenoms respec-
tively in Western blot. Among the therapeutic polyvalent antivenoms,
VB was found to be relatively better reactive/cross-reactive with
V. russellii, E. carinatus and N. naja venoms than BSV as evidenced by
thick cross reactivity bands. BSV and VB both showed very little cross-
reactivity with O. hannah venom nearly to a similar extent. In both the
cases, only one protein band around 43 kD mass showed cross-
reactivity (lane 4, Fig. 3B and C). As expected OH-IgG readily reacted
with O. hannah venom as intense reactivity bands were seen throughout
the protein distribution range in the gel (Fig. 3D, lane 4).
4.2. In vitro neutralization of enzymatic activities of O. hannah venom
by antivenoms
Both BSV and VB polyvalent antivenoms did not neutralize the pro-
tease and indirect hemolytic activities, but at the venom to OH-IgG ra-
tios above 1:10 (w/w), only VB showed marginal inhibition of
 Y.J. Gowtham et al. / International Immunopharmacology 21 (2014) 148–155 151

Fig. 1. Cross-reactivity of snake venoms with antivenoms in Ouchterlony immuno double diffusion assay: Central wells contain 400 μg each of antivenoms BSV (A), VB (B) and OH-IgG (C).
Peripheral wells 1, 2, 3 and 4 respectively contain 200 μg each of O. hannah, N. naja, V. russellii and E. carinatus venoms in all the cases.

hyaluronidase activity. While, OH-IgG effectively neutralized the prote-
ase (Fig. 4A), hyaluronidase (Fig. 4B) and indirect hemolytic (Fig. 4C) ac-
tivities of O. hannah venom. The inhibition was dose dependent in all
the cases. At the venom to OH-IgG ratios (w/w) of 1:7, 1:6, and 1:5,
50% inhibition while, at 1:12, 1:10 and 1:14 ratios, complete inhibition
of protease, hyaluronidase and indirect hemolytic activities were ob-
served respectively. Among the activities, indirect hemolytic activity
was inhibited least.
4.3. In vitro neutralization of toxicity of O. hannah venom by antivenoms
Fig. 5 depicts the effect of antivenoms on re-calcification time of
citrated human plasma. BSV and VB both antivenoms marginally
inhibited the pro-coagulant activity, VB caused little more inhibition
over BSV. In contrast, OH-IgG effectively inhibited the pro-coagulant ac-
tivity dose dependently. At the venom to OH-IgG ratios (w/w) of about
1:5 and 1:9, 50% and 100% inhibition was observed respectively. The

Fig. 2. Cross-reactivity of snake venoms with antivenoms in ELISA: Antivenoms from BSV (A), VB (B), and OH-IgG (C) were used. The antivenoms were diluted in PBS from independent
stocks of 180 μg/100 μl PBS. Values represent mean ± SEM of three experiments.
152 Y.J. Gowtham et al. / International Immunopharmacology 21 (2014) 148–155

Fig. 3. Cross-reactivity of snake venoms with antivenoms in Western blot assay: SDS-PAGE (10%) was carried out and the gels were respectively treated with Coomassie brilliant blue R250
(A), BSV (B), VB (C) and OH-IgG (D) antivenoms. In all the cases, lanes 1 to 4 were loaded respectively with V. russellii, E. carinatus, N. naja and O. hannah venoms. M represents molecular
weight markers in kD (rabbit muscle myosin (205), phosphorylase B (97.4), bovine serum albumin (66), ovalbumin (43), carbonic anhydrase (29), soyabean trypsin inhibitor (20.1), ly-
sozyme (14.3), aprotinin (6.5), and insulin (3.5) under non-reduced condition).

Fig. 4. Inhibition of enzyme activities. A: Protease, B: hyaluronidase: and C: indirect hemolytic activity: O. hannah venom 0.2 mg each for protease (A) and hyaluronidase (B), and 0.05 mg
for indirect hemolytic (C) activities were pre-incubated with increasing amounts (w/w) of BSV, VB and OH-IgG antivenoms. Values represent mean ± SEM of three experiments.
 Y.J. Gowtham et al. / International Immunopharmacology 21 (2014) 148–155 153

Fig. 5. Inhibition of pro-coagulant activity: O. hannah venom (40 μg) was pre-incubated
with increasing amounts (w/w) of BSV, VB and OH-IgG antivenoms. Values represent
mean ± SEM (n = 3), p b 0.01.

citrated plasma revealed the re-calcification time of 4.5 ± 0.03 min
which served as control, while the antivenoms did not affect the clotting
time of plasma. Similarly, BSV and VB antivenoms marginally reduced
the O. hannah venom caused elevated levels of serum creatine phospho-
kinase (CPK) and lactate dehydrogenase (LDH) activities; however, OH-
IgG caused effective and dose dependent reduction (Fig. 6A and B). The
control serum levels of CPK and LDH were found to be 292 ± 12 and
1126 ± 34 respectively. At the venom to OH-IgG ratios (w/w) of 1:8
and 1:11, 50% reduction and at 1:13 and 1:15 complete reduction of
CPK and LDH levels that were similar to control values were achieved
respectively. Further, OH-IgG antivenom effectively and dose depen-
dently neutralized the edema inducing activity of O. hannah venom. At
venom: OH-IgG ratio (w/w) of 1:9, the OH-IgG completely inhibited
the edema inducing property as the edema ratios were in accordance
with the control values. In contrast, BSV and VB polyvalent antivenoms
showed very little or no inhibition of edema formation by the venom.
However, the antivenoms themselves did not induce any edema in ex-
perimental mice (Fig. 7). The lethal dose50 (LD50) of O. hannah venom
Fig. 7. Inhibition of edema inducing activity: O. hannah venom (20 μg) was pre-incubated
with increasing amounts (w/w) of BSV, VB & OH-IgG antivenoms. Values are expressed as
mean ± SEM (n = 3), P b 0.01 when compared with controls.
was found to be 2.63 ± 0.33 mg/kg body weight of mice. At 2 LD50, the
death time recorded was 22 ± 0.50 min. At the venom to OH-IgG ratio
(w/w) of 1:15, the death time was extended to 19 h 40 ± 5.25 min. How-
ever, at a ratio of 1:16, complete neutralization of the lethality was
achieved. On the contrary, the therapeutic polyvalent antivenoms (BSV
and VB) could not neutralize the lethality of the venom even at a ratio
of 1:35 venom:antivenom (Table 1).
5. Discussion
In this study, a total of three antivenoms were tested against four
snake venoms for reactivity/cross-reactivity and neutralization effica-
cies. Of the three, BSV and VB are locally available therapeutic polyva-
lent antivenoms from different firms while, OH-IgG is a monovalent

Fig. 6. Inhibition of myotoxicity: The serum creatine phosphokinase (A) and lactate dehydrogenase (B) activities. The O. hannah venom (LD25) was pre-incubated with increas-
ing amounts (w/w) of BSV, VB and OH-IgG antivenoms. Values are expressed as IU/L. Values of VB and BSV represent mean ± SEM (n = 3), p b 0.05 and values of OH-IgG rep-
resent mean ± SEM (n = 3), p b 0.001.
154 Y.J. Gowtham et al. / International Immunopharmacology 21 (2014) 148–155
Table 1
Inhibition of lethality: O. hannah venom (2 LD50) was pre-incubated independently with
increasing amounts of BSV, VB & OH-IgG antivenoms and injected intraperitoneally to
groups of mice and the survival time was recorded for 24 h.
Venom:antivenom BSV VB OH-IgG
ratios (w/w)
1: N.P N.P N.P
1:10 N.P N.P N.P
1:14 N.P N.P Death time
extended
1:16 N.P N.P Survived
1:20 N.P N.P Survived
1:25 N.P N.P survived
1:35 Death time Death time Survived
extended extended
Values represent mean ± SEM of three individual experiments when P b 0.01. NP = no
protection.
antivenom which was prepared against O. hannah venom in rabbit.
Ouchterlony immunodouble diffusion, ELISA and Western blot assays
greatly vary with respect to their sensitivity. ELISA being the most sen-
sitive assay can detect picograms of antigens, the determined titer value
of 16,500 for OH-antiserum suggests the moderate antigenic nature of
O. hannah venom. However, several factors could contribute for the ef-
ficacy of immune response and the response may even vary among in-
dividual animals as well. Despite, O. hannah and N. naja are Elapid
snakes and their venoms are rich in highly toxic phospholipases and
three finger toxins, OH-IgG showed moderate cross-reactivity with the
venom of the later. The observed cross-reactivity is because of the less
toxic high molecular mass (approximately above 40 kD) venom toxins
but not for the low molecular mass (approximately 6 to 12 kD) toxins
which includes the highly toxic phospholipases and three finger toxins
as no much detectable cross-reactivity bands are seen in the molecular
mass range around 6 to 12 kD in Western blot analysis (Fig. 3D lane 3).
This suggests the lack of adequate numbers of cross-reacting antigenic
determinants/epitopes among the toxic phospholipases and three fin-
ger toxins of the venoms; hence the cross-neutralization of toxic prop-
erties of the venoms is a matter of concern. Further, both BSV and VB
being polyvalent in nature prepared for the venoms from different spe-
cies, they did not reveal any signs of cross-reactivity bands with the
O. hannah venom in the molecular mass region where phospholipases
and three finger toxins would appear in Western blot. In this study,
Western blot and ELISA techniques could pick the cross-reactivity sig-
nals of OH-IgG and as well as BSV and VB better while Ouchterlony
immunodouble diffusion technique failed to detect. The observed better
reactivity/cross-reactivity of VB over BSV could be because of the rea-
sons including the immunological endurance of the animals used for
immunization, dosage of cocktail of venoms injected and as well as
venom variability.
The surface epitopes not only confer the antigenicity but also reflect
on the structural features of various toxins in their native form present
in snake venoms. The least or no cross-reactivity of BSV and VB with
O. hannah venom toxins clearly suggests that the toxins present in
these venoms appear to vary in their antigenic properties or antigenic
determinants and hence likely in their structures. In all the neutraliza-
tion experiments, both BSV and VB did not show any signs of measur-
able neutralization of any of the enzymatic and toxic properties
studied. However, OH-IgG monovalent antivenom effectively neutral-
ized all enzymatic and toxic properties of O. hannah venom. For exam-
ple, protease, hyaluronidase and indirect hemolytic activities were not
neutralized by both BSV and VB, while OH-IgG did neutralize effectively.
The surface epitopes not only confer the antigenicity but also reflect on
the structural features of various toxins in their native form present in
snake venoms. The least or no cross-reactivity of BSV and VB with
O. hannah venom toxins might suggest that the toxins present in these
venoms might vary in their antigenic properties or antigenic determi-
nants and hence likely in their structures. Further, immunological
response for an antigen may vary among species and among individuals
of the same species suggesting the complex nature. In venom toxins, the
most toxic toxin may not be most antigenic and also, it is likely that an-
tigenicity and toxicity of toxins may not go together. In addition, as
snake venoms show tremendous variations in composition, the relative
abundance of venom toxins may vary. These factors could have affected
the quality of BSV and VB which are polyvalent in nature prepared for
the mixture of venoms from ‘big four’ snakes as compared to OH-IgG
which was made only for O. hannah venom.
It is quite interesting that the snake venoms studied here although
target the same vital functions to kill or to immobilize the prey or
human victims differ markedly in their antigenicity and hence likely in
their structures and therefore their mode of target action as well. Al-
though venom neutralization by antivenom in snakebite cases is a mat-
ter of great concern, the treatment efficacy invariably depends on the
dose of venom injected, the time gap in between the bite and the start
of treatment, and most importantly the efficacy of the antivenom used
for treatment. Thus, this study has made an attempt for the first time
to establish the neutralizing efficacies of locally available therapeutic
polyvalent antivenoms from BSV and VB that were prepared for the
venoms of big four snakes against king cobra venom so that attempts
can be made to treat accidental king cobra bites. The results of cross-
reactivity, neutralization of enzymatic and toxicity studies confirmed
the absolute inefficacy of BSV and VB antivenoms to neutralize king
cobra venom. However, the monovalent antivenom OH-IgG prepared
in rabbit for king cobra venom readily and effectively neutralized the
enzymatic and toxic properties of king cobra venom.
In conclusion, in the absence of any therapeutic antivenom available
as of now to treat the possible accidental king cobra bite, the results of
this study ruled out the likely use of BSV and VB antivenoms to treat
king cobra bite and hence offer scope for making specific therapeutic
antivenom that can neutralize king cobra venom which can be used
readily to treat the accidental king cobra bite in regions where the
king cobra is endemic in the Indian subcontinent.
Acknowledgment
The authors thank the University Grants Commission, New Delhi,
India (F-36-275/2008 [S.R.] dated 26-03-2009) for financial support.
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