4.12. Double-Immunodiffusion Assay (Ouchterlony Test)

For analyzing the antigens and antibodies this immunological technique was applied [41]. Briefly, 1 g of agarose powder was dissolved in 100 mL of sterile 1× PBS in a glass beaker (final concentration of 1%) followed by heating at 100 °C for 10 min to achieve a homogeneous solution. 0.5 mg of sodium azide was then added as antimicrobial agent to prevent unwanted bacterial growth. The solution was slowly poured onto the plastic plate on a horizontal surface and waited 30 min for solution to solidify and form flat gel. The gel was then punched with disposable vacuum plastic pipette to form a series of apart holes in it. 50 µL of mice anti-toxoid sera was placed in the middle hole, 50 µg of rPLD1 in the adjacent hole, 50 µg of mutant-rPLD1 in the other adjacent hole, the next one 500 µg of crude venom (PLD consists 10% of the whole crude venom) and the last one was filled with 100 µL of PBS solution as negative control. After antisera and other antigens were absorbed into the gel during the initial 15 min, all the holes refilled three times with the respective antisera and antigen solutions for obtaining more precise result. The plate was then covered with its lid and kept in humidified chamber (a box with wet cotton) on a horizontal surface at 37 °C for 24 h. This test was performed for both antisera of mice immunized by Freund’s adjuvant and alum adjuvant.