Document Type : Original Article
Authors
1
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran;
3
Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
4
Department of Pathology, Iran University of Medical Sciences, Tehran, Iran
5
Student Research Committee, Department of Immunology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
6
Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran
10.22034/iji.2026.111414.3211
Abstract
Background: High quality antibodies are essential for reliable immunohistochemical detection of tumor biomarkers. Human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and GATA binding protein 3 (GATA3) are widely used markers in diagnostic pathology. Objective: To generate and characterize novel monoclonal antibodies (mAbs) targeting human HER2, CK5/6, and GATA3 antigens and evaluate their IHC performance versus commercial antibodies.
Methods: Short peptide sequences derived from HER2, CK5/6, and GATA3 were conjugated to keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice. Hybridoma technology was employed to generate mAbs, which were screened by ELISA and IHC. Selected clones were expanded and mAbs were purified by affinity chromatography. Antibody characteristics were assessed by ELISA, Western blotting, and flow cytometry. IHC performance was evaluated on human tissue specimens and compared with commercial diagnostic antibodies. Concordance and diagnostic performance were assessed using Cohen’s kappa, Spearman’s correlation, and Bland–Altman analysis.
Results: Monoclonal antibodies against HER2, GATA3, and CK5/6 were generated. Clones 1E4 (HER2), 2D5 (GATA3), and 1B4 (CK5/6) were selected, with affinity constants of 0.8, 15.7, and 1.9 nM, respectively. Immunohistochemical analysis demonstrated high concordance with commercial antibodies (97.3–100%), and Bland–Altman analysis confirmed agreement between the generated and commercial antibodies. All selected clones showed 100% specificity, while sensitivities were 93.5% for clone 1E4 and 100% for clones 2D5 and 1B4.
Conclusion: The generated mAbs demonstrated diagnostic performance comparable to the commercial antibodies for immunohistochemical detection of HER2, GATA3, and CK5/6, supporting their potential as reliable alternatives for diagnostic applications.
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