Blood samples were collected 1 h later and serum creatine kinase (CK) activity was measured using Merck Granutest 2.5. Concentrations of 0, 25, 50, and 100 μg of purified 59/2-E4 mAb were incubated with 5 μg of B. atrox venom and injected i.d. into the shaved back of three Swiss mice. After 30 min, animals were euthanized and the size and intensity of subcutaneous hemorrhage
in injected areas was estimated. 3.5 mg samples of purified mAb 6AD2-G5 were preincubated with 150 μg of venom for 30 min at ambient temperature and i.p. injected into five Swiss mice (18–22 g). One hour after inoculation, the tips of tails were cut and immersed in 10 mL of distilled water until bleeding stopped (Assafim et al. 2006; Greene et al. 2010). learn more The optical density of samples was determined in a spectrophotometer at 410 nm. In addition, 500 μL of horse F(ab′)2 bothropic antivenom was used as positive control group, whereas venom plus saline was injected into the mice as negative control. Groups of five Swiss mice (18–20 g) were injected i.p. with selleck chemicals 500 μL saline containing 5 mg 59/2-E4, 5 mg A85/9-4, and 3.5 mg 6AD2-G5 mAb. After 30 min, mice were challenged s.c. with 350 μg of crude venom. Controls were injected i.p. with 500 μL saline and challenged s.c. with 350 μg
of venom. In another experiment 10.5 mg of mAbs (3.5 mg of each mAbs) were incubated with 200 μg of venom for 30 min at 37 °C followed i.p. injection into the mice. The control group received 200 μg of venom. Survival/death rates were recorded at 24 and 48 h. A mixture containing 3.45 mg each of mAb 59/2-E4, A85/9-4, and 6AD2-G5 incubated with 200 μg of venom was injected i.p. in groups of six Swiss mice. Controls received only saline and venom. After 2, 24, and 48 h, two mice from each group were euthanized by CO2 inhalation and their tissues and organs removed and fixed in 10% neutral p-formaldehyde. Tissues were dehydrated in ascending concentrations of ethanol (70–100%) and embedded in paraffin
using an automatic tissue processor (TP 1020, Leica, Germany). Docetaxel in vitro Then, 5 μm sections were stained with hematoxylin-eosin and tissue sections were observed using a digital image analysis system coupled to a microscope (Zeiss axioplan/axiocam, Germany). We evaluated the lethality neutralization by monoclonal antibodies against three major toxic components of B. atrox venom to test the prospects of developing bothropic antivenom based on monoclonal antibodies. General features of purified mAb specific to serineproteinase (thrombin-like 6AD2-G5 clone), PLA2 (A85/9-4 clone), and hemorrhagin (Zn-metalloproteinase 59/2-E4 clone) are shown in Fig. 1. When submitted to SDS-PAGE analysis, all three mAb preparations demonstrated two major protein bands, one of around 55 kDa and one of approximately 29 kDa, suggestive of immunoglobulin heavy and light chains, in addition to several minor contaminant bands ( Fig. 1A).