Background: Cytokeratin 7 (CK7) and cytokeratin 20 (CK20) are widely used immunohistochemical (IHC) markers routinely employed to delineate the primary site of epithelial malignancies, especially when the primary tumor is unknown. However, access to high-quality commercial antibodies remains limited in many laboratory settings.
Objective: To establish a practical and reproducible workflow to generate mouse-derived polyclonal antibodies against human CK7 and CK20.
Methods: Recombinant full-length proteins and epitope-enriched fragments were expressed in Escherichia coli, purified, and used for immunization. Antibody responses were evaluated by ELISA, while specificity and diagnostic applicability were assessed using Western blot and IHC on formalin-fixed, paraffin-embedded tissue sections. Post-production refinement, including protein A purification and negative absorption, was applied to reduce non-specific binding and improve antibody performance.
Results: High-titer antibody responses were successfully generated, with epitope-based antigens eliciting stronger and more selective reactivity compared to full-length proteins. Initial antibody preparations showed broad staining across epithelial tissues, which is expected given the conserved nature of cytokeratin. However, purification and absorption steps significantly reduced non-specific staining and improved specificity. In IHC analyses, the generated antibodies demonstrated staining patterns consistent with established CK7/CK20 expression profiles and were comparable in interpretability to commercially available reagents.
Conclusion: This study demonstrates that diagnostically usable polyclonal antibodies against human CK7 and CK20 can be produced using a cost-effective and accessible approach. This workflow provides a practical alternative for laboratories with limited access to commercial antibodies and supports the continued use of IHC as a reliable tool for tumor classification.