Maad Rayan Publishing Company Biomedicine Advances 3080-0382 2 4 2025 10 20 Development of a sensitive, reliable, and cost-effective sandwich ELISA for quantitative measurement of human hemoglobin A2 in beta-thalassemia diagnosis 195 199 10.34172/bma.35 EN Safura Pakizehkar https://orcid.org/0000-0002-8935-4686 Samaneh Hosseinzadeh Seyedeh Adeleh Razavi Hoda Gholab Ghadaksaz Laleh Hoghooghi Rad Mehdi Hedayati https://orcid.org/0000-0001-5816-775X Journal Article 10.34172/bma.35 2025 06 07 2025 08 02 Introduction: Accurate quantification of hemoglobin A2 (HbA2) is essential for the diagnosis and monitoring of beta-thalassemia and related hemoglobinopathies. Existing methods, such as HPLC and capillary electrophoresis (CE), are effective but require expensive instrumentation. We developed and validated a cost-effective and sensitive sandwich ELISA for measuring HbA2 levels in human blood samples. Methods: A pair of specific monoclonal antibodies against distinct epitopes of HbA2 was used to establish a sandwich ELISA. The assay was optimized for analytical performance, including sensitivity, specificity, linearity, and intra-/inter-assay precision. Validation was conducted using both standard recombinant HbA2 and clinical samples from healthy donors and confirmed beta-thalassemia patients. Findings: The assay demonstrated a lower limit of detection (LOD) of 0.1% and a quantification range of 0.1–10%. Coefficients of variation (CVs) were below 10% across all concentrations tested. HbA2 levels in beta-thalassemia patients were significantly elevated (P<0.001) compared to healthy controls, confirming the assay’s diagnostic potential. Conclusion: This optimized sandwich ELISA offers a reliable, affordable, and scalable alternative for quantifying HbA2 in clinical laboratories, particularly in resource-limited settings. Its high specificity and reproducibility make it a promising tool for the diagnosis and follow-up of beta-thalassemia.