The polyclonal antibody functionality produced in rabbits against Francisella orientalis was evaluated using the double gel immunodiffusion technique (Ouchterlony). This method involves the deposition of antibodies and antigens into wells in an agar gel, allowing diffusion and interaction, resulting in a precipitation line when a specific reaction occurs. This enables the assessment of antibody presence and specificity without the need for additional staining.
Slides were prepared with 1% agar in PBS, with a central well for the rabbit polyclonal antibody and peripheral wells for different antigens: F. orientalis protein extracts at concentrations of 0.462 mg/mL, 0.047 mg/mL, and 0.010 mg/mL, as well as whole inactivated bacteria without disruption. The slides were incubated in a humid chamber at 37 °C for 48–120 hours.
Results showed that the precipitation line was observed only with the 0.462 mg/mL protein extract, indicating a positive and specific reaction between the polyclonal antibody and the antigen. No precipitation line was observed with the lower concentrations or with whole bacteria, suggesting that either the antigen quantity was insufficient to elicit a detectable reaction, or the native antigen was not accessible for interaction.
These findings confirm the polyclonal antibody specifically recognizes the F. orientalis protein extract, demonstrating its functionality and potential applicability in diagnostic assays and infection monitoring, providing a solid foundation for subsequent immunological testing.