3-1. Preparation of Venom Solution
Electroshock lyophilized H. lepturus venom was purchased from Razi Institute (Ahwaz, Iran). The venom was dissolved in normal saline solution to a final concentration of 1 mg/ml and immediately stored at 4°C until needed.
3-2. Preparation of RBC Suspensions
Three 10-ml aliquots of heparinized human blood samples, donated by 3 volunteers, were used for preparing the RBC suspensions. A 2-ml aliquot of each sample was mixed with 6 ml of normal saline and centrifuged for 3 min at 3000 g. This process was repeated 3 times to ensure the removal of all blood plasma. The washed RBC suspension was finally diluted with normal saline to 5% concentration and stored at 4°C until assayed.
3-3. Assessment of the Hemolytic Efficacy of the Venom
The aim of this series of experiments was to assess the hemolytic efficacy of the venom (Razi Institute, Karaj, Iran) and select a standard concentration for later experiments. For this purpose, triplicate Eppendorf® tubes for each of the 3 blood samples, containing 0.5 ml of RBC suspension and varying concentrations (2, 10, 20, and 40 µg/ml) of the venom, were incubated at 37°C. For the measurement of spontaneous hemolysis, control RBC samples were maintained under the same experimental conditions in the absence of venom. After varying periods (30 min and 2, 4, 8, 12, and 24 h) of incubation, the tubes were centrifuged at 14000 rpm for 3 min (Eppendorf centrifuge model 5410, Germany). A 100-µl sample of each supernatant was transferred to a 96-well plate and hemolysis was measured using a spectrophotometric plate reader (Sunrise plate reader; Tecan, Austria) by reading absorbance at 450 nm.
3-4. Test of Antivenom Activity
In this series of experiments, the effectiveness of antivenom in inhibiting hemolysis was assessed under 2 different experimental conditions:
3-5. Test of Direct Neutralizing Capacity of the Antivenom
In order to evaluate the efficacy of the antivenom in inhibiting hemolysis, 20 µg/ml of venom, an amount found to produce obvious hemolysis after 2 h of exposure, was mixed with 0%, 4%, 10%, or 20% (v/v) antivenom and allowed to stand for 5 min. Then, 0.5 ml of RBC suspension was added to the mixture. After centrifugation to pellet the intact RBCs, the optical density of supernatant samples was measured as described in section 3-3, following the same incubation periods. The degree of hemolysis, i.e., the change in optical density, was compared between the untreated and venom-treated RBC samples at the corresponding periods of incubation.
3-6. Test of the Efficacy of Antivenom in the Presence of Washed RBCs
In order to assess the effect of RBCs on the efficacy of the antivenom, i.e., its specificity, 0.5 ml of RBC suspension was mixed with different concentrations of antivenom (4%, 10%, or 20% v/v) and allowed to stand for 5 min. Then, 20 µg/ml of the venom was added to each sample and incubated as described in section 3-3. Hemolysis was measured and compared with both an untreated negative control and a positive control containing 20 µg/ml of venom only. In order to assess any possible hemolytic activity of the antivenom, a separate series of experiments was conducted: similar concentrations (4%, 10%, or 20% v/v) of antivenom were mixed with 0.5 ml of RBC suspension and incubated as described in section 3-3. The resultant changes in hemolysis were compared with an untreated control.
3-7. Statistical Analysis
The results were expressed as mean ± SEM. The significance of differences between the means for different treatment groups was assessed using ANOVA followed by Duncan,s test for multiple comparisons. Values of P < 0.05 were considered significant.