A Novel Accurate Rapid ELISA for Detection of Urinary Connective Tissue Growth Factor, a Biomarker of Chronic Allograft Nephropathy

Abstract

Background Chronic allograft nephropathy (CAN) is a leading cause of kidney graft failure. The latest evidence suggests that connective tissue growth factor (CTGF) may be a biomarker of CAN. Detection of urinary CTGF levels is a potential noninvasive strategy to predict the early onset of CAN. Compared to the traditional “sandwich” enzyme-linked immunosorbent assay (ELISA), we established a novel, accurate, faster, one-step competitive indirect ELISA (Ci-ELISA) to estimate the urinary CTGF concentrations in humans, rats, and mice. Materials and Methods We used recombinant human CTGF, which is an 11.2 kDa protein of 98 amino acid residues containing the C-terminal portion of the full-length CTGF protein having 96% and 94% homology with rats and mice, as the coating antigen and standard competitor. We then applied polyclonal antibody against the CTGF C-terminal portion, secondary antibody conjugated with horseradish peroxidase (HRP), and urine samples to establish one-step Ci-ELISA. Rat kidney allograft recipients were sacrificed at 4, 8, or 12 weeks posttransplantation. We harvested serum, urine, and transplanted kidneys. Hematoxylin-eosin (H&E), periodic acid-Schiff (PAS), and Masson trichrome stains were used to estimate the histopathologic changes according to the Banff schema. Serum creatinine (SCr) levels were measured to represent renal function. CTGF in kidney was detected by immunohistochemistry. Urinary CTGF concentrations were measured using the one-step Ci-ELISA. Results The whole one-step Ci-ELISA test can be accomplished within 2 hours. The detection limit of the assay was 0.16 ng/mL CTGF. The time-dependent elevated recipient concentration of urinary CTGF was positively correlated with SCr, histologic changes, and CTGF in the kidney after transplantation. The 95% confidence intervals of urinary CTGF concentrations in control rats were 24.4 to 35.2 ng/mL; at 4, 8, and 12 weeks posttransplantation they were 139.4 to 395.6, 826.7 to 1429.5, and 3206.0 to 4448.2 ng/mL, respectively. Conclusions A rapid one-step Ci-ELISA was successfully established to detect the CTGF levels in urine. These results further indicated that detection of urinary CTGF is a potential noninvasive strategy to predict the early onset of CAN.