Structural Characterization and Immunogenic Evaluation of a 50 kDa Thyroid Peroxidase Fraction Isolated from Hypothyroid Patient Serum

Devi Ramadani (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Aulanni'am Aulanni'am (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Muh. Fikry Baharuddin (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Almas Dwi Khairana (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Saidun Fiddaroini (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Muhammad Fikri Nur (Brawijaya University)
Hilman Nurmahdi (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Wibi Riawan (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Arie Srihardyastutie (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Akhmad Sabarudin (Department of Chemistry, Faculty of Mathematics and Natural Science, Brawijaya University, Malang, 65145, Indonesia)
Dyah Kinasih Wuragil (Faculty of Veterinary Medicine, Brawijaya University, Malang, East Java, 65145, Indonesia)
Rulli Rosandi (Metabolic and Endocrinology Division, Department of Internal Medicine of Saiful Anwar Hospital, Brawijaya University, Malang, East Java, 65145, Indonesia)
Achmad Rudijanto (Endocrinology Divison, Department of Pediatrics of Saiful Anwar General Hospital, Brawijaya University, Malang, East Java, 65145, Indonesia)
Ahmad Lubab (Department of Mathematics, Faculty of Science And Technology, Sunan Ampel State Islamic University, Surabaya, East Java, 65145, Indonesia)

Science and Technology Indonesia
Vol. 11 No. 4 (2026): October

Thyroid peroxidase (TPO) is a key enzyme involved in thyroid hormone biosynthesis and one of the major autoantigens in autoimmune thyroid disease. However, studies investigating immunoreactive serum-derived TPO-related protein fractions remain limited. This study aimed to isolate an approximately 50 kDa immunoreactive protein fraction from the serum of hypothyroid patients, characterize its biochemical properties, and evaluate its immunogenicity for rabbit polyclonal antibody production. Serum samples were initially characterized based on anti-TPO antibody levels and total protein concentration. Rabbits were immunized with the isolated protein, and the resulting immune response was evaluated by indirect ELISA, SDS-PAGE, Western blotting, histopathology, and immunohistochemistry. SDS-PAGE consistently revealed a distinct protein band at approximately 50 kDa. ATR-FTIR analysis demonstrated characteristic protein-associated functional groups, while LC-MS/MS confirmed the proteinaceous nature of the isolated fraction through amino acid composition analysis. Immunization induced a progressive increase in anti-TPO IgG levels, and the purified rabbit IgG exhibited the expected electrophoretic profile under reducing and non-reducing conditions. Western blot analysis demonstrated that the generated polyclonal antibodies recognized the isolated approximately 50 kDa protein fraction, whereas no signal was detected in the secondary antibody-only control. Histopathological and immunohistochemical analyses further supported activation of the immune response following immunization. These findings demonstrate that the isolated approximately 50 kDa immunoreactive protein fraction possesses biochemical and immunogenic properties and has potential as an experimental immunogen for polyclonal antibody production.