Abstract

Background:
Toxoplasmosis is a zoonotic disease caused by the parasite Toxoplasma gondii (T. gondii), which can infect any warm-blooded intermediate host, including humans. Dense Granule Protein 7 (GRA7), which possesses high immunogenicity, is one of the potential T. gondii antigen candidates for diagnostic development.

Aim:
This study aimed to develop polyclonal antibodies specific to the recombinant GRA7 protein and evaluate their detection capability.

Methods:
Two male New Zealand White rabbits were immunized with recombinant GRA7 protein using a primary immunization protocol, followed by two booster doses on days 14 and 28. Serum samples were collected on days 14, 21, 28, 35, and 42 to analyze antibody levels using ELISA, while antibody specificity was tested using Western blot.

Results:
ELISA analysis demonstrated a significant increase in optical density (OD) values by day 28 (0.519, 1.050, and 1.067), followed by a pronounced peak at day 42 (1.480, 1.511, and 1.547) across all tested antigen concentrations (1, 5, and 10 µg/mL). These findings indicate the development of a robust and sustained humoral immune response. Western blot analysis confirmed specific recognition of the recombinant GRA7 protein at approximately 63 kDa, with protein bands appearing thicker and more distinct in the 10⁻⁶ dilution. This indicated that antibodies at the 10⁻⁶ dilution have a higher concentration, enabling a stronger signal in Western blot visualization, reflecting a high-affinity antibody response.

Conclusion:
The polyclonal antibodies generated against the recombinant GRA7 protein demonstrated strong preliminary immunoreactivity and high binding capacity, supporting their potential application as key components in the development of immunological diagnostic tools for toxoplasmosis. Further specificity assays are required to improve the performance of these newly produced antibodies.

Key words: GRA7, Polyclonal Antibodies, Toxoplasma gondii, Western Blot, Zoonoses