Snake venom
Venom was extracted from specimens of wild-caught large brown spitting cobras (Naja ashei) maintained at the Bioken Snake Farm, Watamu, Kenya. Table S1. Collected venom was snap-frozen and stored at -20°C. Reconstitution was done in phosphate-buffered saline at the time of use.
Antivenom
The antivenoms used in this study are described in Table S2 and were acquired from hospitals in Kisumu County, Kenya.
Protein content determination of the venom and antivenoms
The protein content of Naja ashei venom and test antivenoms was estimated by Lowry’s method by preparing serial dilutions of bovine serum albumin in triplicate and pipetting 0.2 ml of each dilution into 10 ml glass tubes. Two ml of alkaline copper sulphate was added to each glass tube which was then incubated at room temperature for 10 minutes before 0.2 ml of Folin-Ciocalteau reagent was added to the tubes. A 30 minute incubation time was allowed and the absorbance values of the dilutions were recorded at 660 nm on a Spectronic 21D UV-Visible spectrophotometer [10]. A standard curve was used to infer the protein content in venom and antivenoms. Table S3.
SvPLA2 activity of Naja ashei venom
The svPLA2 activity of Naja ashei venom was determined by the methods of Haberman and Hardt 1972 and Felix Silva and colleagues [11, 12] with modifications. A 1:3 v/v ratio of egg yolk and phosphate-buffered saline was prepared. 90 ml of phosphate-buffered saline was added to 10 ml of the 1:3 v/v egg yolk suspension and the mixture was added to 300 ml of a 1% w/v agarose solution. Finally, 125 µl of 0.1mM Calcium chloride was added to the resulting mixture which was then poured into sterile Petri dishes in a laminar airflow cabinet to solidify. Six mm wells were made on the agarose-egg yolk media and 10 µl of serially diluted venom (0.5-22.5 µg/ml) was pre-incubated at 37°C for 60 minutes in 1 ml Eppendorf tubes, pipetted into the 6 mm wells on the agarose-egg yolk media, and incubated for 24 hours at 50°C. A 1:10, v/v solution of Carbol Fuchsin was used to visualize the svPLA2 induced halos. Vernier calipers were used for measurement. Figure S2. Phosphate-buffered saline was used as a negative control. All experiments were carried out in triplicate and the minimum phospholipase concentration (MPC50) of the venom was calculated using regression analysis.
Neutralization of the svPLA2 activity of Naja ashei venom by antivenom
The method of Iwanaga and Suzuki 1979 was used with modifications. [13]. Figure S3. 10 µl of a 2×MPC50 dose of N. ashei venom was mixed with 20 µl of various doses (25-400 µg/ml) of antivenom I and II in a 96-well ELISA plate by use of a microplate shaker for 5 minutes. The plate was further incubated at 37°C for 20 minutes. 200 µl of the substrate (1.1% egg yolk suspension in 0.1M PBS adjusted to pH 8.1 and 125 µl 0.2mM CaCl2) was added to the 96-well plate which was subsequently incubated at room temperature and the change in absorbance of the substrate from 0 to 30 minutes was measured spectrophotometrically on a multi-plate reader at 620 nm [13]. Figure S3. All experiments were performed in triplicate.
Brine shrimp lethality of Naja ashei venom
The method of Meyer and colleagues was used [14]. Ten, 48-hour old brine shrimp larvae were transferred from a hatching trough to 5 ml sample vials by use of Pasteur pipettes. Aliquots (5, 50 and 500 µl) of 5 mg/ml stock solutions of the venoms were pipetted into the 5 ml sample vials which were made up to the mark with a 38.5% w/v marine salt solution so that the final concentration of the venoms was 10, 100, and 100 µg/ml respectively. Phosphate-buffered saline was used as control and surviving larvae were counted after 24, 48, and 72 hours. All experiments were performed in quintuples and probit analysis was used to determine the median lethal concentration responsible for 50% mortality of brine shrimp after 24, 48, and 72 hours [15–17]. Figure S4. Meyer’s and Clarkson’s toxicity indices were used to classify venom toxicity/lethality [18, 19].
Neutralization of Naja ashei induced brine shrimp lethality by antivenom
The WHO (World Health Organization) protocol on venom neutralization by antivenoms was used with modifications [3]. Various doses of the antivenoms (25-400 µl of 100 mg/ml) were mixed with a 2LC50 dose of N. ashei venom. Figure S4. This dose corresponds to a dose that is lethal to 100 % of the brine shrimp larvae. The venom/antivenom mixtures were incubated at 37°C for 30 minutes and added to 5 ml sample vials that contained brine shrimp larvae. The number of surviving larvae in each group was recorded after 24 hours. The median effective concentration of antivenom was calculated using probit analysis.