Abstract
Abstract Background: 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 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. Materials and 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. Results: The assay demonstrated a lower limit of detection (LOD) of 0.1% and a quantification range of 0.1–10%. Coefficients of variations (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.