Fig 1: Specific aptamers obtained after multiple rounds of the SELEX process. a The binding rate of 1 μg of NGAL protein during every SELEX round, and the binding rate of NGAL protein increased from the initial 0.079% at round 1 to 1.89% at round 7. b The binding status between 53 aptamers and NGAL protein were primarily validated using the quantitative method, and ΔCt greater than 4 served as the selection standard. c The binding amounts of candidate aptamers, including NA10, NA36, NA42, NA53 and NA21 to NGAL-coated magnetic beads are much higher than those of bare magnetic beads using the qPCR method (n = 3, for each group, *p < 0.05, **p < 0.01, ***p < 0.001 vs control, and the error bars indicate the mean ± standard deviation SD)
Fig 2: The performance verification of the ELAA method established with NA53 aptamers. a The ELAA method with NA53 aptamers can detect NGAL without cross-reaction with the albumin and globulin after specificity analysis. b The establishment of standard curve and the detection range of the ELAA method. c The sensitivity analysis of the ELAA method with NA53 aptamers
Fig 3: The analysis of the specificity and affinity between the candidate aptamers and NGAL protein. a The specificity assay of the binding status between aptamers and bare magnetic beads, NGAL-coated beads, and normal human serum-coated beads using the qPCR method. b The Kd value of candidate aptamers was analysed after serial dilution, and NA53 aptamers possess the strongest binding capability on NGAL proteins. c NGAL protein and NA53 are colocalised in oesophageal squamous carcinoma cells KYSE450 using immunohistochemistry. Herein the green channel represents the interactions between aptamer library or NA53 and NGAL protein, the red channel represents the interactions between NGAL antibody and NGAL protein, and the blue channel represents the occurrence of nuclear after staining with DAPI, and the merged channel represents the overlap of green channel, red channel and blue channel
Fig 4: Schematic illustration of the ELAA method established in this study. The ELAA detection method was established using the strategy of sandwich structure of antibody-NGAL-NA53 aptamers
Fig 5: Laboratory finding of patients with sepsis, healthy controls and patients with rheumatic disease.Patients with sepsis (n = 15) are presented with black, healthy controls (n = 8) before inflammatory stimulus by exercise with white open bars and after stimulus with white striped bars and patients with rheumatic disease (n = 23) before treatment with an anti-TNF agent with grey open bars and after treatment with grey striped bars. (A) Plasma-NGAL levels, (B) plasma creatinine levels (Crea), (C) estimated glomerular filtration rate (eGFR-EPI), (D) leukocyte count in whole blood, (E) plasma interleukin 6 levels (IL-6), (F) plasma interleukin 8 levels (IL-8), (G) plasma interleukin 10 levels (IL-10) and (H) plasma levels of C-reactive protein (CRP) are presented. * P<0.05, ** P<0.01, *** P<0.001 compared to sepsis or indicated by clamp to corresponding group. Cytokine detection limits were 2.5 pg/ml for IL-6, 3.6 pg/ml for IL-8 and 3.3 pg/ml for IL-10. Bars and error bars represent median values and interquartile range.
Supplier Page from Sino Biological, Inc. for Human LCN2 / NGAL Protein (His Tag)