Production and Characterization of Novel Murine Monoclonal Antibodies Against Programmed Death-Ligand 1 With Potential Diagnostic Application in Immunohistochemistry Publisher Pubmed



Mansouri M ; Mohammadi M ; Montazer F ; Faghih ZS ; Soltanshahi M ; Shokri F ; Jedditehrani M ; Shabani M
Source: Analytical Biochemistry Published:2026

Abstract

Background: Programmed death-ligand 1 (PD-L1) is one of the main immunoregulatory proteins. It binds to its receptor, PD-1, on the surface of T cells and suppresses their anti-tumor activity. Therefore, elevated levels of PD-L1 protein could predict a potential benefit from PD-L1/PD-1 checkpoint inhibitor immunotherapy. Immunohistochemistry (IHC) is the current standard technique for assessing PD-L1 expression in tumor tissues, and a sensitive and specific monoclonal antibody (mAb) to PD-L1 is a critical parameter for accurate results. Thus, in the current study, we aimed to produce new anti-PD-L1 mAbs with potential diagnostic applications in IHC. Methods: By immunizing mice with human recombinant PD-L1 protein, specific mAbs were produced via the hybridoma method and then screened using enzyme-linked immunosorbent assay (ELISA) and IHC techniques in parallel. Furthermore, isotypes and affinity constants of the selected and purified mAbs were determined, and their specificities were evaluated in Western blot, flow cytometry, IHC, and immunocytochemistry (ICC) assays. Afterward, their reactivity was compared with commercially available anti-PD-L1 mAb in optimized IHC using human placenta tissue and 54 paraffin-embedded bladder cancer tissue samples, and the analytical sensitivity, specificity, and accuracy of homemade IHC were assessed. Results: Among 30 clones that produce significant amounts of specific antibodies against PD-L1 in ELISA, three anti-PD-L1 mAbs (clones 1D6, 1D8, and 1F8) demonstrated high reactivity to their target antigen in human placental and cancerous tissues in IHC. Moreover, one of the selected mAbs, clone 1D6, was also able to specifically recognize its target by flow cytometry, western blotting, and ICC using a panel of human PD-L1 positive and negative tumor cell lines and placenta tissue. Compared to commercially available anti-PD-L1, the analytical sensitivity, specificity, and accuracy calculated for clone 1D6 were 94.87 %, 80 %, and 91.83 %, and for clone 1D8, they were 97.67 %, 81.81 %, and 94.4 %, respectively. Conclusion: This study's results demonstrated that the novel anti-PD-L1 mAbs could recognize the target antigen with high specificity and sensitivity. Therefore, they might be appropriate tools for research and diagnosis. © 2026 Elsevier Inc.