Proteolytic Inhibition Assays

Anti-Fibrinolytic

  • The minimal amount of venom that causes a 5 mm clearing zone on a fibrin plate is determined.

  • Equal amounts of two minimal fibrinolytic doses (MFD) are then incubated with various two-fold serial dilutions of the three antivenins.

  • The antifibrinolytic dose is defined as the amount (µg) of antivenin inhibiting the degradation of fibrin by one MFD.

  • The lower the number the more efficient the antivenin.

Anti-Hide Powder Assay

  • The minimal amount of venom that causes an absorbance reading of 0.1 at 595 nm is determined using hide powder azure.

  • Equal amounts of two minimal hide powder doses (MHPD) are then incubated with various two-fold serial dilutions of antivenins.

  • The antihide powder azure dose is defined as the amount (µg) of antivenin inhibiting an absorbance reading of 0.1 of one MHPD of each of the venoms.

    • The lower the number the more efficient the antivenin.

Anti-Gelatinase Assay

  • The minimal amount of venom that causes a clear zone on a Kodak X-OMAT scientific imaging film with gelatin coating is determined.

  • Equal amounts of two minimal gelatinase doses (MGD) are then incubated with various two-fold serial dilutions of the three antivenins.

  • The antigelatinase dose is defined as the amount (µg) of antivenin inhibiting the clearance of the gelatin on the X-ray film by one MGD of venom.

  • The lower the number, the more efficient the antivenin.

Inhibition of venom proteolytic activity on B chain of insulin by antivenoms or animal sera using Capillary electrophoresis (CE)

  • A Beckman Capillary electrophoresis P/ACE 5500 is used to observe the inhibition of venom activity by antivenom or animal sera (Sánchez et al., 2001).

  • Ten microliters of a 1 mg/ml venom or venom fraction are incubated with 10 µl of a 1 mg/ml of antivenom or animal sera for 1 hr at room temperature.

  • After 1 hr, 10 µl of the venom/serum or antivenom mixture is added to 10 µl of a 4 mg/ml of B chain of insulin.

  • The mixture is immediately separated for 10 min at 20 kV, 19.5 µamps, using a 0.01M Borate buffer, pH 8.3 through a 75 µm I.D. X 50 cm (100 x 800 aperture) free zone capillary.

  • A P/ACE UV absorbance detector at 214 nm is used to detect the peptides.