INTRODUCTION: Claudin-3 (CLDN3), a tight junction protein, regulates cell-to-cell interactions in epithelial or endothelial cell sheets. During tumorigenesis, epithelial cells are transformed, and tumor cells proliferate through out-of-plane division, resulting in external exposure of CLDN3. Since alterations of CLDN3 expression are associated with cancer progression and higher CLDN3 expression is observed in most ovarian cancers, we tested the feasibility of using a CLDN3-specific antibody as a novel imaging tracer. MATERIALS AND METHODS: After reducing the CLDN3-specific antibodies to expose the -SH groups, click chemistry was used to conjugate the radioactive isotope 111In or the fluorescent protein FNR648. Human ovarian cancer OVCAR-3 and glioblastoma U87MG cells were used as CLDN3-positive and -negative cells. Flow cytometry was used to determine the CLDN3 IgG1 monoclonal antibody binding to both cell lines. OVCAR-3 cells were injected subcutaneously into mice to establish a xenograft model. 111In-labeled CLDN3 antibodies (370 kBq/50 µL) were administered intravenously into mice. After 24 h, organs, including tumors, were excised and measured with a γ-counter. Images were acquired with the IVIS optical imaging system and SPECT/CT. RESULTS: The labeling efficiency of NOTA-111In and antibody-NOTA-111In was 98.52% and 100%, respectively. FNR648-labeled CLDN3 antibody bound to the cell surface of OVCAR-3 and U87MG with 83.4% and 5.7% specificity, respectively. In OVCAR-3 tumor xenografted mice, CLDN3 IgG1 antibody showed a 2.5-fold higher tumor uptake (20.4 ± 7.4% ID/g) than human IgG1 (8.8 ± 2.6% ID/g) at 24 h post injection. The CLDN3 antibody fluorescence signal in the tumor peaked at 24 h post injection. CONCLUSION: We have successfully conjugated a radioisotope and a fluorescent protein with CLDN3-specific antibodies and verified the specific binding of labeled antibodies to OVCAR-3 tumors in a mouse model. Our data suggested that CLDN3-specific human monoclonal antibodies could be used as a useful theranostic tracer.