An Antibody-Aptamer-Hybrid Lateral Flow Assay for Detection of CXCL9 in Antibody-Mediated Rejection after Kidney Transplantation

Abstract

Chronic antibody-mediated rejection (AMR) is a key limiting factor for the clinical outcome of a kidney transplantation (Ktx), where early diagnosis and therapeutic intervention is needed. This study describes the identification of the biomarker CXC-motif chemokine ligand (CXCL) 9 as an indicator for AMR and presents a new aptamer-antibody-hybrid lateral flow assay (hybrid-LFA) for detection in urine. Biomarker evaluation included two independent cohorts of kidney transplant recipients (KTRs) from a protocol biopsy program and used subgroup comparisons according to BANFF-classifications. Plasma, urine and biopsy lysate samples were analyzed with a Luminex-based multiplex assay. The CXCL9-specific hybrid-LFA was developed based upon a specific rat antibody immobilized on a nitrocellulose-membrane and the coupling of a CXCL9-binding aptamer to gold nanoparticles. LFA performance was assessed according to receiver operating characteristic (ROC) analysis. Among 15 high-scored biomarkers according to a neural network analysis, significantly higher levels of CXCL9 were found in plasma and urine and biopsy lysates of KTRs with biopsy-proven AMR. The newly developed hybrid-LFA reached a sensitivity and specificity of 71% and an AUC of 0.79 for CXCL9. This point-of-care-test (POCT) improves early diagnosis-making in AMR after Ktx, especially in KTRs with undetermined status of donor-specific HLA-antibodies.

Identification and validation of urinary CXCL9 as a biomarker for diagnosis of acute interstitial nephritis

“…Importantly, the greater concentration of CXCL9 portends improved laboratory test characteristics, and CXCL9 has the potential to be included in existing platforms in clinical laboratories or developed as a point-of-care test. In fact, point-of-care devices identifying CXCL9 are currently under development for diagnosis of acute cell-mediated transplant rejection, which, similar to AIN, is a tubulointerstitial immune-mediated process that spares the glomeruli, and such devices could be adapted for rapid, bedside diagnosis of AIN ( 30 ). Our findings also provide insight into AIN pathogenesis.…”

Monitoring of Serological, Cellular and Genomic Biomarkers in Transplantation, Computational Prediction Models and Role of Cell-Free DNA in Transplant Outcome

“…Another study [ 24 ] concludes that uNGAL may be more helpful in assessing renal function in the first week after KT when combined with other markers. Another study found that NGAL levels in urine and serum could be used to predict kidney damage and as a biomarker of acute kidney injury after transplantation [ 28 ]. According to the researchers, patients with acute kidney injury had higher uNGAL levels than patients without acute kidney injury, with a 2 h post-transplant increase.…”

Aptamer-Based Point-of-Care Devices: Emerging Technologies and Integration of Computational Methods

“…In the literature, this sensing strategy has also been combined with duplex aptamers, where the labelled duplex aptamers were disassociated from their complementary In addition, some studies were designed to combine antibodies and aptamers in a single hybrid LFA. In such an example, CXC-motif chemokine ligand (CXCL) 9 (a critical urinal biomarker for antibody-mediated rejection of kidney transplantation) was detected on a tracking sandwich format LFA [57]. Antibodies specific to CXCL 9 were initially utilized as capture probes on the test line, and the detection aptamers specific to CXCL 9 were conjugated to AuNPs to ensure signal transduction.…”