APTAMERS IN VARIOUS BIOASSAYS
4.2 Materials and Methods
4.2.1 Dot Blot assay using aptamer candidates
A dot blot assay was performed to check the specificity and affinity of selected aptamers.
A 0.5mg/ml stock solution of purified recombinant EGFR ECD was serially diluted 10 fold. A volume of 5µl of protein was spotted on 0.45 μm pore-size NC membrane and air dried. The immobilization of protein was visualized by Ponceau staining. The membrane was blocked with 5% BSA in PBST overnight at 4°C with gentle shaking.
After blocking membrane was treated with 500nM bioaptamer EGFR_A15 and EGFR_A13 for 1hr at RT. After three washing steps, HRP conjugated streptavidin (1:1000 dilution from 1mg/ml stock) was added for 1hr. Color was developed by adding DAB (3, 3’-Diaminobenzidine) substrate. The biotinylated initial aptamer pool were used as a negative control. The biotinylation of non-enriched aptamer pool were performed by PCR using biotinylated Apt_F1 and unlabelled Apt_R1. The sense strand was obtained as described in section 3.2.4.
Figure 4.1: Schematic overview of Dot Blot assay.
4.2.2 In vitro cytotoxicity Assay of aptamer candidates against cancer cell line
Cells were counted and tested for viability before experiment. MDA MB 231 cells were seeded 24 hours prior to experiments in 96-well flat-bottom microtiter plates (Nunc) at a density of 1.0×104 cells/well in DMEM medium containing 10% FBS. Different concentration of selected aptamers and initial aptamer library (25nM, 50nM, 75nM,
100nM, 200nM and 500nM) were added to the cells in DMEM incomplete medium and incubated at 37°C for 24 and 48 hours. The viability of cells were subsequently determined by adding 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) in DMEM incomplete media for 4 hours at 37°C. Formazan crystals were dissolved by adding DMSO. Absorbance was measured at λ=570 nm with background subtraction at 630 nm (Tecan Microplate reader, CA, USA). The cell viability was calculated with the following equation:
Cell viability % = AT / AC × 100
Where AT and AC are the absorbance of aptamer treated and untreated cells, respectively.
4.2.3 In vitro Scratch Assay of aptamer candidates against cancer cell lines
MDA MB 231 cells were grown to a postconfluent multilayer in 24-well plates and serum-starved for 24 hr in the presence of 0–200 nM of either the aptamer or the control (initial pool). A 200 µl pipette tip was used to make a vertical-line scratch. Migration inside the scratch was stimulated by addition of media containing 10% fetal bovine serum and cells were allowed to migrate for 8 hrs. The closing of scratch due to the migration of cells was monitored by microscopy. Cells were fixed with 4% formaldehyde and stained with crystal violet dye. Three independent experiments were performed in duplicate each.
4.2.4 Development of indirect ELAA for the detection of EGFR protein
The indirect ELAA (Enzyme Linked Aptamer Assay) was developed for the sensitive detection of Epidermal Growth factor receptor protein. The development of any enzyme linked immuno assay system involves the optimization of reagents. In other words, the working concentration of each component of the test must be assessed.
4.2.4.1 Optimization of Assay condition
The sensitivity of any assay are dependent on the interaction between biorecognition probe and analyte. Thus prior to the DNA aptamer assay, the assay parameters were investigated for the optimized method. The optimization was carried out using chessboard or checkerboard titrations (CBT). Both biotinylated DNA aptamers
EGFR_A15 and EGFR_A13 were diluted to 1nM, 5nM, 20nM and 50nM. The stock protein solution were serially diluted.
4.2.4.2 Enzyme Linked aptamer assay for EGFR protein
The process of CBT involves the dilution of two reagents against each other to analyse the optimal concentration. The 96 well Maxisorp flat-bottomed polystyrene immuno plates (Nunc, Denmark) were used. The recombinant protein solution were diluted from column 1 to 11, while in the last column i.e. column 12 only diluent were added (Fig.
4.2). The microplate were then incubated overnight at 4° C with gentle shaking for the immobilization of proteins. After washing, blocking of plates were carried out by adding 200 µl of 5% BSA for 1 hour at RT. The plates were extensively washed with PBST buffer to remove unbound BSA. The diluted biotin labelled aptamers were added in different rows and incubated for 60 min at RT. After washing, the plates were incubated with HRP conjugated streptavidin (New Englands Biolabs). The microplates were again washed four times with PBST, the substrate TMB (Sigma) was added and incubated for 10 min at RT. The reaction was stopped by adding 2M HCl and read at 450 nm. A dose response curve was obtained based on these data using non-linear regression analysis in GraphPad Prism 5.0.
Figure 4.2: Plate layout for Checkerboard test for indirect Enzyme Linked Aptamer Assay.
The developed indirect Enzyme Linked aptamer assay using the lead aptamer sequences were also performed on complex samples of EGFR. Cell lysate of A431, MDA MB 231
and Jurkat cell lines were coated on to the microtiter plates and incubated separately with biotin labelled 1nm of EGFR_A15, 20 nM of EGFR_A13 and anti EGFR antibody for 1hr at RT. The wells were washed with PBST buffer five times after incubation. Then Binding and detection reaction were carried out in a similar way as described above.
4.2.4.3 Establishing Indirect Competitive Enzyme Linked aptamer assay for EGFR protein
Microplates (96 wells) were coated with recombinant EGFR ECD protein as described above. The plates were washed with PBST three times and blocked with 200 μL of 5%
BSA at RT for 2 h. Next, a 50-μl aliquot of excess EGFR specific monoclonal antibody at a fixed concentration (1:200 dilution in PBS) was added per well to ensure saturation of all binding sites. 50µl of 1nM Bio-EGFR_A15 was also added to the wells containing antibodies. Similarly in another assay 20nM of Bio-EGFR_A13 alongwith anti EGFR antibodies were added (Fig. 4.3). In last two rows only 50 μL of anti EGFR antibodies was added into the wells as a control. The plates were well mixed on a shaker and incubated at RT for 1 h. Plates were then developed as described above.
Figure 4.3: Schematic representation of indirect competitive ELAA. Anti EGFR antibody and bioaptamer are added to the blocked plate at the same time. Antibodies
compete to inhibit the bioaptamer from binding to the coated EGFR protein, resulting in a lighter colorimetric detection. Adapted with permission from Fu et al., 2014.
4.2.5 Serum Recovery Experiment
4.2.5.1 Isolation of Serum from human blood
Real serum samples were collected from healthy volunteers following an established protocol and ethical guideline. In an anticoagulant free collection tube, 12 ml of blood was collected and allowed to settle for 15-30 mins to clot. The samples were then centrifuged at 2000 g for 10 mins in a refrigerated tube to remove the clot. The supernatant collected was designated as serum and stored at -80°C till further use.
4.2.5.2 Detection of EGFR in spiked serum using selected aptamers
A stock solution of recombinant EGFR ECD protein was serially diluted with serum solution. Each 50µl of EGFR ECD solution diluted in serum and PBS was added to the well. The plates were incubated overnight for the immobilization of proteins. After extensive washing and blocking with 5% BSA, plates were probed with the best recognising biotinylated aptamer sequence i.e. EGFR_A15. 1nM of EGFR_A15 aptamers and then each wells were developed following the same steps described above.
Further the percent recoveries of each concentration were calculated.