Development of the point-of-care test quantifying nephrin excretion
Modified systematic evolution of ligands by exponential enrichment (SELEX)
The advanced SELEX was used as described by Gold et al. Briefly, we prepared a DNA library containing 40-nucleotide randomized region in which dT is substituted with 5-(N-benzyl carboxyamide)-2’-deoxyuridine (Bz-dU) or 5-(N-naphthylcarboxyamide)-2’-deoxyuridine (Nap-dU). The randomized central region was flanked by two conserved regions of 17 nucleotides (5’-GAG TGACCGTCTGCCTG-40N-CAGCCACACCACCAGCC-3’). Twenty-five thermal cycles (93℃ for 30 s, 52℃ for 20 s, and 72℃ for 60 s) were conducted to amplify the library. The library was applied for the SELEX process and the process was performed at 37℃ with following steps. A mixture of 1 mmol of aptamer library dissolved in the buffer (40 mM HEPES/pH 7.5, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.002% Tween 20) was heated at 95℃ for 3 min, and then slowly cooled to 37℃ at 0.1℃/s for re-folding. To eliminate the non-specific binder aptamers, the aptamer library solution was pre-incubated with His-tagged magnetic bead (Invitrogen, Grand Island, NY), and supernatant was collected. The aptamers in supernatant were incubated with 10 pmol of nephrin for 30 min and then the nephrin was captured through the His.
Binding affinity
The aptamer-protein equilibrium dissociation constants (Kd) were determined by the nitrocellulose-filter binding method. Before the binding assay, aptamers were dephosphorylated using alkaline phosphatase and their 5’-ends were radiolabeled using T4 polynucleotide kinase (New England Biolabs) and [32P]-ATP (Amersham Pharmacia Biotech, Piscataway, NJ). The direct binding assays were conducted by incubation of 10pM of 32P-labeled aptamers with recombinant nephrin at a concentration ranging from 1 mM to 10 fM in the selection buffer at 37℃. The fraction of aptamers bound to nephrin was quantified with a PhosPhorImager (Fuji FLA-5100 Image Analyzer, Tokyo, Japan). The data was corrected by subtraction of nonspecific binding signal generated by binding of radiolabeled aptamers to the nitrocellulose filter from the obtained signal.
Binding competition assay
To select the aptamer pairs binding different regions of nephrin, binding competition assays were performed using [32P]-labeled aptamer (hot aptamers) and unlabeled aptamers (cold aptamers). We tested all aptamer pair candidates to identify the best pair that did not compete with each other for binding nephrin. Hot aptamers (2,000 cpm) and 25 pmole cold aptamers were dissolved in S buffer (30 μL) and the solution was heated at 95℃ for 3 min and then slowly cooled to 25℃ at a rate of 0.1℃/s. After transferring to a 96 well plate containing 30 μL of 10 nM nephrin, the aptamer solution was incubated at 25℃ for 15 min. Then, 5.5 μL of Zorbax resin solution was added to the reaction mixture followed agitation in a Thermomixer for 1 min at 1,300 rpm. The mixture was applied to a PVDF filter plate, and the hot aptamers bound to nephrin were quantified using a phosphor-imager (Fuji FLA-5100 Imamge Analyzer).
Preparation of capture and detection aptamers for LFA
The aptamers were modified to be used as capture or detection aptamers. For capture aptamers, 5’ biotin-labelled aptamers were conjugated with neutravidin to efficiently spot them on the nitrocellulose membrane. The biotin-labelled aptamers were dissolved in 5 μL of 1xPBS buffer at the concentration of 80 μM. The solution was heated at 95℃ for 5 min and cooled at 37℃ for 15 min, then mixed with 80 pmol of neutravidin (5 μL) and incubated at 37℃ for 1 hour. The detection aptamers were prepared by conjugating with streptavidin-gold nano particle (SA-GNP). The biotin-labelled aptamers were dissolved in 100 μL of PBS buffer at a concentration of 2 μM and the solution underwent heating and cooling as mentioned above. The solution was mixed with 100 μL of SA-GNP (10 O.D units/mL) and then 250 μL of 1xPBS was added. The mixture was incubated for 1 hour at room temperature with shaking, them the mixture was centrifuge at 4℃, 8,500 rpm. After removing the supernatant, the pellet was resuspended with 100 uL of 1xPBS buffer. We applied 6 uL of the detection aptamer solution for each strip.
Preparation of test strips
The test strip consisted of a sample pad, a nitrocellulose membrane, and an absorbent pad. A 0.8 mm sample pad was immersed in a sample pad solution (10 mM sodium phosphate (pH 7.0), 10% Tween 20) and dried at room temperature in a desiccator. To assemble the test strip, a nitrocellulose membrane was attached to the middle of the backing card, and then a sample pad and an absorbent pad were attached to the bottom and the top of the backing card, respectively. We spotted 0.5 μL of capture aptamer solution and the control aptamer solution onto a nitrocellulose membrane strip and dried for 2 days in room temperature. The schematic illustration of the LFA system using a pair of nephrin specific aptamers is described in figure 1.
Lateral flow assay
We prepared 100 μL of each sample in test tubes and mixed it with 6 μL of detection aptamer solution and 1 μL of DxSO4. Then the samples were soaked via the sample pad on a test strip. We read the signal after 15 mins of incubation and imaged it with a scanner. To determine the limit of detection of LFA for nephrin, we tested with commercially available nephrin. Various concentrations of nephrin (0, 0.5, 1, 2, 5 μg/mL) were prepared by spiking in the LF running buffer or urine. Figure 2 demonstrates the competition of aptamers for binding to nephrin protein. Positive signals were observed from nephrin protein diluted from 5 μg/mL to 0.5 μg/mL. The limit of detection (LOD) of the aptamer-based LFA was 0.5 μg/mL or lower.