Fig 1.
SDS-PAGE and densitogram of venoms in a 10% gel.
Venoms were treated in reducing and non-reducing buffer prior to loading before electrophoresed and the gel was stained using silver staining (A). M indicates the protein marker lane, NS indicates N. sumatrana venom, NK indicates N. kaouthia venom, (R) indicates venoms treated with reducing sample buffer, (N) indicates venoms treated with non- reducing sample buffer, (i) indicates reduced Malaysian N. sumatrana venom, (ii) indicates non-reduced Malaysian N.sumatrana venom, (iii) indicates reduced Malaysian N. kaouthia venom, (iv) indicates non-reduced Malaysian N. kaouthia venom. Densitograms were generated using ImageJ software and the relative mobility for protein band (Rf) corresponding to the peak was manually calculated.
Fig 2.
Comparison of SDS-PAGE and western blot profiling of reduced venoms.
(i) N. sumatrana and: (ii) N. kaouthia venoms (10 μg) were separated in a 10% gel, blotted into a PVDF membrane and visualized using a chemiluminescence substrate. Panel (A) SDS- PAGE profile of venoms. Panel (B-D) are blot membrane that were incubated with Thai Neuro Polyvalent Antivenom, Thai Monocled Cobra Antivenom, and Thai King Cobra Antivenom, respectively. M is lane for protein molecular weight marker.
Fig 3.
Densitogram for western blot of Malaysian Naja sumatrana and Naja kaouthia venoms.
Panels (A-C) are desnsitograms for membrane blot of N. sumatrana venom that were incubated with Neuro Polyvalent Antivenom (NPAV), Monocled Cobra Antivenom (CAV) and King Cobra Antivenom (KCAV),respectively. Panels (D-E) are desnsitograms for membrane blot of N. kaouthia venom that were incubated with Neuro Polyvalent Antivenom (NPAV), Monocled Cobra Antivenom (CAV) and King Cobra Antivenom (KCAV), respectively NS(R) indicates reduced N. sumatrana venom and NK(R) indicates reduced N. kaouthia venom. Densitograms were generated using ImageJ software and the relative mobility for protein band (Rf) corresponding to the peak was manually calculated.
Fig 4.
Neurotoxic effects of Malaysian N. sumatrana venom and N. kaouthia venom in indirectly stimulated twitches of the chick biventer cervicis nerve-muscle preparation.
Panels (A) and (B) indicate change of indirectly stimulated twitch height in tissues exposed in different concentration (5–30 μg/ml) of N. sumatrana and N. kaouthia venoms, respectively. Panels (C) and (D) indicate change in the contractile response to exogenous agonist in tissue exposed to different concentration (5–30 μg/ml) of N. sumatrana and N. kaouthia venoms, respectively. NS indicates N.sumatrana venom and NK indicates N.kaouthia venom * p < 0.05, significantly different from control (distilled water) (n = 3, one-way ANOVA).
Fig 5.
Effect of pre-incubation of antivenoms to indirectly stimulated twitches and contractile responses to exogenous agonists in tissue exposed to Malaysian N.sumatrana and N.kaouthia.
Panels (A) and (B) indicate change in twitch height for tissues that were pre-incubated with recommended titer of Cobra Antivenom (CAV), Neuro Polyvalent Antivenom (NPAV) and King Cobra Antivenom (KCAV) and exposed to N.sumatrana and N.kaouthia venoms (5 μg/ml), respectively. Panels (C) and (D) indicate contractile responses to exogenous agonists in tissues that were pre-incubated with antivenoms after exposure to N.sumatrana and N.kaouthia venoms (5 μg/ml), respectively. NS indicates N.sumatrana venom and NK indicates N.kaouthia venom. * p < 0.05, significantly different than the respective venom alone (n = 3, one-way ANOVA).
Fig 6.
The effect of the addition of antivenom at the t90 time point after exposure to Malaysian N. sumatrana venom and N. kaouthia venom to the indirectly stimulated twitches and response to exogenous agonists.
Panels (A) and (B) venom indicate change of indirect twitches when recommended titer of Cobra Antivenom (CAV), Neuro Polyvalent Antivenom (NPAV) and King Cobra Antivenom (KCAV) was added at t90 point after addition of 5 μg/ml N. sumatrana venom and N. kaouthia venom respectively. Panels (C) and (D) venom indicate contractile responses to exogenous agonists in tissues that were added with recommended titer of Cobra Antivenom (CAV), Neuro Polyvalent Antivenom (NPAV) or King Cobra Antivenom (KCAV) at the t90 time point after addition of 5 μg/ml N. sumatrana and N. kaouthia venoms, respectively. NS indicates N.sumatrana and NK indicates N.kaouthia * p < 0.05, significantly different from venom alone (n = 3, one-way ANOVA).
Fig 7.
Myotoxic effects of Malaysian N. sumatrana venom and N. kaouthia venom in directly stimulated twitches of the chick biventer cervicis nerve-muscle preparation.
Panel (A) N.sumatrana venom and (B) N.kaouthia venom indicate change in the direct twitch height in tissue added with different concentration (5–30 μg/ml) venom. Panel (C) N. sumatrana venom and (D) Absolute baseline change of tissues after addition of (C) N. sumatrana venom and (D) N. kaouthia venom while on direct twitches. Panel (E) N. sumatrana venom and (F) N. kaouthia venom indicate contractile response change to exogenous KCl in tissues exposed to on contractile responses to exogenous KCl. NS indicates N.sumatrana venom and NK indicates N.kaouthia venom.* p < 0.05, significantly different from control (distilled water) (n = 4, one-way ANOVA).
Fig 8.
Effect of pre-incubation with antivenom on directly stimulated twitches of tissues and exposed to Malaysian N.sumatrana and N.kaouthia.
Panel (A) N. sumatrana venom (10 μg/mL) and (B) N. kaouthia venom (10 μg/mL) indicate change of direct twitch height in tissues with prior addition of recommended titer of Cobra Antivenom (CAV), Neuro Polyvalent Antivenom (NPAV) and King Cobra Antivenom (KCAV). Panel (C) N. sumatrana venom and (D) N. kaouthia venom indicate absolute baseline change of tissues with prior antivenom addition and exposure to venom while on direct simulation. Panel (E) N. sumatrana venom and (F) N. kaouthia venom indicate change in contractile responses to exogenous KCl in tissue with prior addition of recommended titer Cobra Antivenom (CAV), Neuro Polyvalent Antivenom (NPAV) and King Cobra Antivenom (KCAV). NS indicates N.sumatrana venom and NK indicates N.kaouthia venom.* p < 0.05, significantly different than the respective venom alone and # p < 0.05, significantly different than control (distilled water) (n = 3, one-way ANOVA).