Development of a fully automated chemiluminescence immunoassay for urine monomeric laminin-γ2 as a promising diagnostic tool of non-muscle invasive bladder cancer

Abstract

Background: Monomeric laminin-γ2 in urine is a potential biomarker for bladder cancer. However, the current detection system uses an antibody that cannot discriminate between monomeric laminin-γ2 and the heterotrimeric γ2 chain of laminin-332, which may cause false-positive reactions. The present study aimed to develop a fully automated chemiluminescence immunoassay system using a specific monoclonal antibody against monomeric laminin-γ2.

Methods: In total, 237 urine specimens (84 from patients with bladder cancer, 48 from patients with benign urological disease, and 105 from healthy donors) were collected, and monomeric laminin-γ2 values in the urine were measured using a fully automated chemiluminescence immunoassay.

Results: The results revealed that laminin-γ2 values in patients with benign urological disease were comparable to those of healthy donors and that the chemiluminescence immunoassay's lower limit of detection was 10 pg/mL (approximately 20-fold better than the sandwich enzyme-linked immunosorbent assay's limit of 200 pg/mL). Moreover, the chemiluminescence immunoassay demonstrated that patients with bladder cancer, including non-muscle invasive bladder cancer (≤pT1), had higher laminin-γ2 values than patients with benign urological disease or healthy donors.

Conclusions: These results suggest that urine monomeric laminin-γ2 may be a promising biomarker to diagnose cases of non-muscle invasive bladder cancer using a fully automated chemiluminescence immunoassay system.

Keywords: Chemiluminescence immunoassay (CLIA); Monomeric laminin-γ2; Non-muscle invasive bladder cancer (NMIBC); Urine biomarker.

Fig. 1

SPR analysis of the specificity of the 2H2 mAb using a BIAcore 3000. a Sensorgrams for the interactions between the 2H2 monoclonal antibody (mAb) and mono-Ln-γ2 and b between the 2H2 mAb and Ln-332. The 2H2 mAb was immobilized onto the surface of CM5 chips, varying concentrations of mono-Ln-γ2 and Ln-332 were injected, and binding was monitored using surface plasmon resonance. The injected concentrations of mono-Ln-γ2 and Ln-332 were 0, 0.625, 1.25, 2.5, 5.0, 7.5, and 10.0 μg/mL

Fig. 2

Calibration curves. a The standard curve from an ELISA for Ln-γ2 using a conventional Ln-γ2 antibody, the D4B5 mAb, and the DIII pAbs. The detection range was 200–20,000 pg/mL. b The standard curve for the chemiluminescence immunoassay (CLIA) using the 2H2 mAb and the DIII pAbs. The detection range was 10–20,000 pg/mL. The CLIA had 20-fold greater sensitivity compared with the sandwich ELISA. c Standard curve for Ln-332 using the CLIA with an anti-Ln-α3 mAb and the anti-DIII pAbs. The detection range was 100–10,000 pg/mL

Fig. 3

Dot plot analysis of urine mono-Ln-γ2 with BC, benign urological disease, and HD. Dot plots of creatinine-corrected urine Ln-γ2, (Ln-γ2/crn) in patients with urological diseases. a Dot plots of Ln-γ2/crn in 84 patients with bladder cancer (BC), 48 patients with benign disease, and 105 healthy donors (HDs). The standard deviation values for urinary Ln-γ2 were small in patients with benign diseases and HDs. The distributions of urine Ln-γ2/crn values in patients with BC were significantly higher compared with those from patients with benign disease and HDs (p = 0.0006 and p < 0.0001, respectively). b Dot plots of Ln-γ2/crn in 44 patients with non-muscle invasive BC (NMIBC), nine patients with muscle invasive BC (MIBC), and 105 HDs. The distributions of urine Ln-γ2/crn values in the NMIBC and MIBC groups were significantly higher than those from the healthy donors (p < 0.0001)

Fig. 4

Receiver operating characteristic (ROC) curve analysis. a Bladder cancer (BC) vs. benign urological disease and healthy donors (HDs), (b) BC vs. HDs, (c) non-muscle invasive BC (NMIBC; ≤pT1) and muscle invasive BC (MIBC; ≥pT2) vs. HDs, (d) NMIBC vs. HDs, and (e) MIBC vs. HDs. The area under the curve (AUC) value for BC vs. benign disease and HDs (a) was 0.87, and the AUC value for BC vs. HDs (b) was 0.81. These AUC values are sufficient to facilitate a diagnosis of BC. The AUC values for NMIBC and MIBC (c), NMIBC (d), and MIBC (e) were all 0.86. Thus, mono-Ln-γ2 in urine can be used to diagnose both NMIBC and MIBC

Fig. 5

Comparison of mono-Ln-γ2 and Ln-332 values in patients with benign urological diseases and HDs. Dot plots of creatinine-corrected urine Ln-γ2 (Ln-γ2/crn) and Ln-332 (Ln-332/crn) from 41 patients with benign urological diseases and 44 healthy donors. The distributions of urine Ln-332/crn were wider than the distributions of urine Ln-γ2/crn in patients with benign urological diseases and in the healthy donors. These results indicate that Ln-332 is secreted into the urine of patients with benign urological disease and healthy donors, which could lead to false-positive diagnoses