Abstract
BackgroundNeutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker of early diagnosis and prediction for acute kidney injury (AKI). However, the current program for NGAL detection is not extensively applied in clinics due to the high expense of antibodies. Nucleic acid aptamers are single-strand DNAs or RNAs which could bind to targets with high specificity and affinity, and they have been widely used in the diagnosis and therapy for multiple diseases. It is valuable for us to develop a new method for NGAL detection using aptamers instead of antibodies to achieve increased efficiency and decreased cost.MethodsNucleic acid aptamers against NGAL were obtained after SELEX process using magnetic beads, and an enzyme-linked aptamer analysis (ELAA), which can be widely used in clinical diagnosis at low cost, were successfully established. The feasibility of ELAA was further validated with urine samples harvested from 43 AKI patients and 30 healthy people.ResultsThree candidate aptamers, including NA36, NA42 and NA53, were obtained after 8 rounds of SELEX process with magnetic beads and verified by quantitative polymerase chain reaction (qPCR), and the Kd value of each aptamer was 43.59, 66.55 and 32.52 nM, respectively. Moreover, the linear relationship was consistent at the range of 125–4000 ng/mL, and the detection limit of ELAA assay was 30.45 ng/mL. We also found that NGAL could be exclusively detected with NA53, and no cross-reaction between NA53 and human albumin or globulin occurred, the coefficient of variation (CV) between inner-plate and inter-plate was less than 15%, and the recovery rate was between 80 and 110%. Moreover, the sensitivity and specificity of ELAA assay in this study are 100% and 90%, respectively. Consistently, these results could also diagnose whether the occurrence of AKI in lots of patients, which has been demonstrated with the ELAA method we established after using NA53.ConclusionsTaken together, NA53, the best candidate aptamer targeting NGAL protein, can be applied in clinical testing.
Highlights
Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker of early diagnosis and prediction for acute kidney injury (AKI)
Aptamers targeting NGAL are successfully obtained using SELEX process After coating with NGAL protein, the coating efficiency was determined to be up to 91.4% (Additional file 2: Figure S1), namely, 0.21 μg NGAL protein was coated with 1 μL of magnetic beads, and the concentration we used was adjusted to 0.20 μg NGAL/μL of magnetic beads for the process of screening
The sequencing result of NA21 was verified by the quantitative polymerase chain reaction (qPCR) method when comparing with other candidate aptamers NA10, NA36, NA42, and NA53, and we found that NA10 and NA21 were not suitable for further studies due to their low capability of binding to NGAL-coated beads (Fig. 2c)
Summary
Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker of early diagnosis and prediction for acute kidney injury (AKI). Neutrophil gelatinase-associated lipocalin (NGAL), known as lipocalin 2, is a secreted protein possessing 178 amino acids in the form of monomers, homodimers and heterodimers [1], It is widely expressed in neutrophils, kidney, prostate, respiratory and digestive tract epithelial cells [4] and plays a vital role in natural immunity [5], anti-oxidation [6], tumorigenesis and tumour metastasis [7, 8]. Once surgery-related inflammation and renal tubular epithelial cell injury occurred, the concentration of NGAL rapidly increased in blood and urine, and it closely correlated with the AKI severity [9]; it may be served as a powerful indicator in clinical application [10]
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