Urine cytology has poor sensitivity for detecting low-grade bladder cancer (BC), while cystoscopy, though highly accurate, is unsuitable as a screening method due to its invasiveness. As a result, there is growing interest in urinary BC-associated proteins as promising diagnostic biomarkers for BC. EphA2, a protein in the ephrin receptor family, is part of a group of receptor tyrosine kinases. EphA2 plays a crucial role in various cellular processes, including cell growth, migration, and survival. In cancer, EphA2 is often overexpressed, leading to uncontrolled cell signaling that promotes tumor growth and metastasis. Overexpression of EphA2 has been reported in multiple malignancies, including head and neck cancer, colorectal cancer, breast cancer, prostate cancer, and ovarian cancer. However, research on EphA2 in relation to BC remains limited. We developed an anti-EphA2 sandwich ELISA to evaluate the sensitivity of EphA2 detection in urine biopsy.

In the initial phase of clinical testing, we collected urine samples from 19 patients with bladder cancer and 12 individuals without BC. Voided urine samples (>30 ml) were collected on the night before each patient underwent transurethral bladder tumor resection. We used a pair of self-developed monoclonal antibodies that recognize two distinct epitopes on EphA2 to capture free EphA2 in liquid biopsy samples. One antibody was immobilized on a microplate to facilitate sample testing, and an enzyme-labeled second antibody was then used to complete the detection reaction, producing a colorimetric outcome in the ELISA. All patients with BC were histologically diagnosed by experienced pathologists and documented.

After concentrating urine proteins 20-fold, we observed a significant difference between the cancer group (n=19) and healthy controls (n=12) (p<0.001). The assay achieved an AUC of 0.956, with a threshold of 86.7 ng/ml, yielding a sensitivity of 89.5% and a specificity of 91.7%.

We successfully developed a pair of monoclonal antibodies against EphA2 using the phage display system, creating a convenient and high-sensitivity sandwich ELISA detection system for EphA2 in liquid biopsy. For distinguishing BC from non-cancer individuals, the assay achieved an AUC of 0.956, indicating a strong level of diagnostic performance for BC. Large-scale studies are needed to validate the diagnostic potential of urinary EphA2 in BC.