<![CDATA[Congenital hypothyroidism (CH) is among the most prevalent and preventable causes of intellectual disability in neonates worldwide, with thyroid-stimulating hormone (TSH) serving as the primary biochemical screening biomarker. In Indonesia, limited availability of locally produced anti-TSH antibodies restricts the scalability of neonatal CH screening programs. This study aimed to isolate TSH from neonatal umbilical cord serum and to evaluate its capacity to induce polyclonal antibodies suitable for immunoassay-based CH screening. Umbilical cord serum was processed using PBST-PMSF buffer and ethanol precipitation, followed by SDS-PAGE separation. A 28 kDa protein fraction was confirmed as a TSH-enriched antigen by Western blot analysis using a monoclonal anti-TSHβ antibody, then isolated via electroelution for immunogenic assessment. Structural characterization was performed by ATR-FTIR spectroscopy and LC-HRMS amino acid profiling. The electroeluted fraction was subsequently used as an immunogen in New Zealand White rabbits, and antibody performance was evaluated by indirect ELISA, dot blot analysis, splenic histopathology, and immunohistochemistry (IHC). Western blot confirmed a single immunoreactive band at 28 kDa. ATR-FTIR revealed a characteristic absorption at 1046 cm⁻¹, consistent with carbohydrate-associated structures of glycoproteins, while LC-HRMS detected amino acids compatible with TSH subunit composition, including arginine, leucine, lysine, and cystine. Immunization successfully induced polyclonal antibody production, with peak reactivity at a 1:200 serum dilution by indirect ELISA. Dot blot analysis demonstrated optimal antigen-antibody binding at a 1:10 dilution. Splenic histopathology confirmed systemic immune activation, evidenced by significant white pulp enlargement (110.25 ± 11.67 µm vs. 88.47 ± 6.67 µm; P < 0.05). IHC demonstrated specific antibody reactivity localized to thyrotroph cells of the rat anterior pituitary. These findings provide preliminary evidence supporting the utility of neonatal cord serum-derived TSH as an antigenic source for local polyclonal antibody production. Further analytical and clinical validation, including cross-reactivity assessment and diagnostic performance evaluation, is required prior to application in routine CH screening.]]>
