Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 18;17(1):204.
doi: 10.1186/s12967-019-1955-7.

Development of a novel ssDNA aptamer targeting neutrophil gelatinase-associated lipocalin and its application in clinical trials

Xiaoqian Hong  1   2 , Huihui Yan  1 , Fuan Xie  1 , Kaiyu Wang  1 , Qiang Wang  3 , Huijuan Huang  4 , Kunrong Yang  1   5 , Suhong Huang  2 , Tingting Zhao  1   6 , Junkai Wang  7 , Yunyun Chen  1   6 , Kuancan Liu  8   9   10 , Xiaopeng Lan  11
Affiliations

Development of a novel ssDNA aptamer targeting neutrophil gelatinase-associated lipocalin and its application in clinical trials

Xiaoqian Hong et al. J Transl Med. .

Abstract

Background: Neutrophil 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.

Methods: Nucleic 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.

Results: Three 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.

Conclusions: Taken together, NA53, the best candidate aptamer targeting NGAL protein, can be applied in clinical testing.

Keywords: Acute kidney injury; Aptamers; NGAL protein.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The schematic flow for the magnetic SELEX process. Multiple steps were involved in the SELEX process, including ssDNA pool synthesis, negative selection, positive selection, elution, cloning, sequencing and SDS-PAGE separation
Fig. 2
Fig. 2
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. 3
Fig. 3
The predicted secondary structure of NA36, NA42 and NA53 aptamers. a The predicted secondary structure of NA36 aptamers. b The predicted secondary structure of NA42 aptamers. c The predicted secondary structure of NA53 aptamers
Fig. 4
Fig. 4
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. 5
Fig. 5
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. 6
Fig. 6
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. 7
Fig. 7
The ELAA method established with NA53 aptamers can distinguish AKI patients in clinical samples. a NGAL protein level in AKI patients is significantly higher than its level in healthy samples after application of the ELAA method with NA53 aptamers. b NGAL protein diagnostic value and ROC curve analysis. c The level of NGAL protein in urine of 4 AKI patients decreased from the acute phase to recovery phase with the ELAA method, which is consistent with the change of creatinine (each colour in the figure represents one patient with AKI)
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Shang W, Wang Z. The update of NGAL in acute kidney injury. Curr Protein Pept Sci. 2017;18:1211–1217. doi: 10.2174/1389203717666160909125004. - DOI - PubMed
    1. Devarajan P. Neutrophil gelatinase-associated lipocalin: a promising biomarker for human acute kidney injury. Biomark Med. 2010;4:265–280. doi: 10.2217/bmm.10.12. - DOI - PMC - PubMed
    1. Lisowska-Myjak B. Serum and urinary biomarkers of acute kidney injury. Blood Purif. 2010;29:357–365. doi: 10.1159/000309421. - DOI - PubMed
    1. Candido S, Abrams SL, Steelman LS, Lertpiriyapong K, Fitzgerald TL, Martelli AM, Cocco L, Montalto G, Cervello M, Polesel J, et al. Roles of NGAL and MMP-9 in the tumor microenvironment and sensitivity to targeted therapy. Biochimica et Biophysica Acta (BBA) Mol Cell Res. 2016;1863:438–448. doi: 10.1016/j.bbamcr.2015.08.010. - DOI - PubMed
    1. Bellmann-Weiler R, Schroll A, Engl S, Nairz M, Talasz H, Seifert M, Weiss G. Neutrophil gelatinase-associated lipocalin and interleukin-10 regulate intramacrophage Chlamydia pneumoniae replication by modulating intracellular iron homeostasis. Immunobiology. 2013;218:969–978. doi: 10.1016/j.imbio.2012.11.004. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources