Anti-Metalloproteases: Production and Characterization of Polyclonal IgG Anti-F2 Fraction Antibodies Purified from the Venom of the Snake Bitis arietans

Abstract

Bitis arietans is a medically important snake found in Sub-Saharan Africa. The envenomation is characterized by local and systemic effects, and the lack of antivenoms aggravates the treatment. This study aimed to identify venom toxins and develop antitoxins. The F2 fraction obtained from Bitis arietans venom (BaV) demonstrated the presence of several proteins in its composition, including metalloproteases. Titration assays carried out together with the immunization of mice demonstrated the development of anti-F2 fraction antibodies by the animals. The determination of the affinity of antibodies against different Bitis venoms was evaluated, revealing that only BaV had peptides recognized by anti-F2 fraction antibodies. In vivo analyses demonstrated the hemorrhagic capacity of the venom and the effectiveness of the antibodies in inhibiting up to 80% of the hemorrhage and 0% of the lethality caused by BaV. Together, the data indicate: (1) the prevalence of proteins that influence hemostasis and envenomation; (2) the effectiveness of antibodies in inhibiting specific activities of BaV; and (3) isolation and characterization of toxins can become crucial steps in the development of new alternative treatments. Thus, the results obtained help in understanding the envenoming mechanism and may be useful for the study of new complementary therapies.

Keywords: Bitis arietans; antivenoms; metalloproteases; polyclonal antibodies.

Figure 1

BaV chromatographic profile and electrophoretic profiles of F2 fraction and the BaV. (A) Sixty milligrams of lyophilized venom were subjected to a chromatography step on an affinity column, equilibrated, and eluted with 25 mM TRIS, pH 8. Samples were eluted at a continuous flow rate of 1 mL/min, and the protein content was monitored under absorbance at 280 nm on the UPC-900 reader. (B) Five µg of BaV and five µg of the F2 fraction were subjected to gradient gel electrophoresis (5% for the upper gel and 12% for the lower gel) under non-reducing conditions. The bands were revealed by silver nitrate impregnation.

Figure 2

Cross-recognition by ELISA of the venoms of snakes of the genus Bitis against the F2 anti-fraction antibody. Cross-recognition of antibodies against B. arietans, B. gabonica, B. nasicornis and B. rhinoceros venoms was determined by the ELISA method in 96-well plates sensitized with one µg of antigen/well. The anti-F2 fraction antibodies were serially diluted (1:500 to 1:256.000) in PBS/BSA 0.1%. Detection with peroxidase-conjugated “anti-mouse” antibodies was performed at a dilution of 1:5000. The reading was taken with ELX 800 plate spectrophotometer (Biotek Instruments, Vermont, USA) at a wavelength of 490 nm. The yield was presented as ELISA units/mL (EU/mL). The assay was performed in duplicate. Data were statistically analyzed using GraphPad Prism version 7 for Windows (GraphPad Software, San Diego, CA, USA). The yield was presented as units per milliliter. * p < 0.05.

Figure 3

Cross-recognition by immunoblotting of venoms from snakes of the genus Bitis against the anti-F2 fraction antibody. (A) Five µg of each venom and five µg of the F2 fraction were subjected to gradient gel electrophoresis (5% for upper gel and 12% for lower gel) under non-reducing conditions. The bands were revealed by silver nitrate impregnation. (B) The nitrocellulose membrane was incubated for 1 h at room temperature with the anti-F2 fraction antibody diluted 1:200 in PBS/BSA 0.1%. After washing with PBS/Tween-20 0.5%, the membrane was incubated for 1 h at room temperature with the “anti-mouse” IgG antibody conjugated with alkaline phosphatase, diluted 1:5000 in PBS/BSA 0.1%.

Figure 4

Experimental plasma affinity. A 96-well plate was primed with one µg of antigen/well, and the dilution of anti-F2 fraction antibodies was set at 1:1000. KSCN concentration ranged from 0 M to 5 M. The percentage of antibodies bound to 3 M KSCN was used to calculate affinity. (A) F2 affinity curve. (B) raw venom affinity curve. (C) percentage of antibodies bound to KSCN 5M. The assay was performed in duplicate. Data were statistically analyzed using GraphPad Prism version 7 for Windows (GraphPad Software, San Diego, CA, USA). The yield was presented as units per milliliter.

Figure 5

Hemorrhagic activity of BaV. Groups of mice (n = 20) were inoculated by intradermal injection with increasing amounts of BaV or PBS pH 7.2. (A) Hemorrhagic tissue fragments corresponding to 10 µg/animal. (B) Hemorrhagic tissue fragments corresponding to 20 µg/animal. (C) Hemorrhagic tissue fragments corresponding to 30 µg/animal. (D) Fragments of hemorrhagic tissue corresponding to 40 µg/animal. (E) Fragments of hemorrhagic tissue corresponding to PBS pH 7.2 inoculation in control animals. The diameter of the area of each tissue fragment was plotted in the ImageJ 1.8.0 program and expressed in mm².

Figure 6

Serum neutralization of BaV hemorrhagic activity. Groups of mice (n = 16) were inoculated by intradermal injection with the MHD of 10 µg of BaV together with different concentrations of the anti-F2 fraction antibody, or PBS pH 7.2. (A) Hemorrhagic tissue fragments corresponding to 10 µg/animal + anti-F2 fraction antibodies 1:5. (B) Hemorrhagic tissue fragments corresponding to 10 µg/animal + antibodies anti-F2 fraction 1:10. (C) Hemorrhagic tissue fragments corresponding to 10 µg/animal + antibodies anti-F2 fraction 1:20. (D) Hemorrhagic tissue fragments corresponding to the inoculation of 10 µg/animal of BaV in PBS pH 7.2 in control animals. The diameter of the area of each tissue fragment was plotted in the ImageJ 1.8.0 program and expressed in mm².