Team:GreatBay SCIE/Measurement

Measurements


During the whole assay, we set up a series of control experiments seriously, and measure them repeatedly. Finally, we successfully measure the binding curve of the aptamer targeting for the Her2 receptor. Which also proves the validity and repeatability of our measurement.

MB ELONA

This assay employs Magnetic Beads, which is modified with -NH2. This method uses MB to localize proteins for the ligand to bind on. HER2 ECD solution is added to activated-MB, and incubated for 1hr. Then it is placed on a magnetic rack, and the supernatant is removed. It is washed several times to remove unbound protein and biotinylated aptamers of different concentrations are added to incubate with protein-coated MB. The MB is then washed again to remove unbound aptamer and HRP-conjugated streptavidin is added for incubation, followed by washing and the addition of TMB solution. At last, stopping solution is added after incubation. MB is removed from the resulting yellow solution(if positive) and can be tested for absorbance at OD450 under the microplate reader.

Materials

  • MB Coupling Kit from Sangon

    • Magnetic Beads (-COOH)(500 nm)
    • Activation Buffer
    • Coupling Buffer
    • EDC
    • Sulfo-NHS
    • Blocking Buffer
    • Preservation Buffer

  • Aptamer Modification: Biotin Labelling & Chemical Modification

  • HER2 ECD Protein from sinobiological

    • Her2/ERBB2 Protein, Human, Recombinant (ECD, His Tag)

  • PBST (1*PBS + 0.1% Tween 20)

  • PBS Buffer

  • TMB Reagent

  • Streptavidin-HRP

  • Sulphuric Acid at 2M

Coating of HER2 Receptors on Magnetic Beads

Preparation

  1. Take 5 μL of magnetic beads in a clean EP tube, and swirl the magnetic beads with vortex mixer for 15 seconds.

Note: The binding capacity of magnetic beads is 10-100 μg of protein per 1 mg of magnetic beads, the amount of magnetic beads and protein should be calculated in advance.

  1. Place the EP tube on the magnetic separation frame for 30-60s so that the magnetic beads are fixed on the EP tube wall, then discard the supernatant.
  2. Remove the EP tube from the magnetic frame, add 2x (MB volume) of activation buffer; mix thoroughly.
  3. Place the EP tube on the magnetic separation frame for 30-60s so that the magnetic beads are fixed on the EP tube wall, then discard the supernatant.
  4. Repeat steps 3-4 twice.

Coating:

Activation

  1. Prepare 50 mg/mL of EDC and 50 mg/mL of Sulfo-NHS using the activation buffer, respectively.

Note: EDC and Sulfo-NHS should be prepared when using, stored away from light, and kept in ice before use. Activating 1 ml of magnetic beads requires 25 mg of EDC and 25 mg of Sulf- NHS.

  1. Add 1.5x (MB volume) of activation buffer, 0.5x (MB volume) of EDC and 0.5x (MB volume) of Sulfo-NHS into the EP tube containing magnetic beads, mix well and incubate at room temperature for 15 min.
  2. Place the EP tube on the magnetic frame for 30-60s so that the magnetic beads are fixed on the EP tube wall, then remove the supernatant.

Coupling

  1. Take 2.5μg of protein and magnetic beads and mix thoroughly, mix 2x (MB volume) coupling buffer into the EP tube.
  2. Incubate on a rotator at room temperature for 90 min or 4 °C overnight.
  3. Place the EP tube on the magnetic rack for 30-60s so that the magnetic beads are fixed on the EP tube wall, then remove the supernatant.

Blocking

  1. Add 5x (MB volume) of blocking buffer to the EP tube, mix thoroughly and incubate on a rotator at room temperature for 30 min.
  2. Place the EP tube on the magnetic frame for 30-60s so that the magnetic beads are fixed on the EP tube wall, then remove the supernatant.
  3. Add 5x (MB volume) of blocking buffer to the EP tube, mix well and place the EP tube on the magnetic frame for 30-60s so that the magnetic beads are fixed on the EP tube wall, then remove the supernatant.
  4. Add 5x (MB volume) of preservation buffer to the EP tube, mix well and place it on the magnetic frame for 30-60s to fix the magnetic beads on the EP tube wall, then remove the supernatant.
  5. Repeat step 3-4 twice.
  6. Add 1x (MB volume) of preservation buffer to the EP tube, mix thoroughly and store at 2-8℃.

Note

  1. Calculate the number of magnetic beads and protein in advance.
  2. EDC and Sulfo-NHS should be ready for use, kept away from light, and placed on ice before use.
  3. Buffers that contain primary amine (such as Tris and glycine) inhibit the binding of proteins to magnetic beads. If the protein is dissolved in primary amine-containing buffers, use dialysis or desalination to remove the interfering components in this buffer before coupling.
  4. The protein concentration of the reaction needs to be optimized. Too low of a protein concentration will cause the magnetic beads to cross-couple. For expensive antibodies, if the protein concentration is too low, other proteins (such as BSA) can be added to occupy the remaining active sites.

Binding of Aptamer with HER2 ECD

  1. Add 5μL of HER2-MB, swirl gently; Place it on Magnetic Separation Frame for 60s; remove the supernatant.
  2. Dilute Biotin-HER2-Apt to desired concentration by using Binding Buffer (PBS+MgCl2). Place in 90°C metal bath for 5 min, then immediately place into ice for 15 min.
  3. Add 50 μL of diluted aptamer into 5 μL MB.
  4. Incubation at 37°C for 15min, swirl every 5 min, make sure the MB is in full contact with the solution; after 15min, remove the supernatant.
  5. Add 50μL PBST, swirl gently; and place the system on Frame for 60s; remove the supernatant. Repeat a total of 5 times.
  6. Add 100uL of 1/2000 diluted streptavidin-HRP
  7. Incubation at 37°C for 15min, swirl every 5 min, make sure the MB is in full contact with the solution; after 15min, remove the supernatant.
  8. Add 50μL PBST, swirl gently; and place the system on Frame for 60s; remove the supernatant. Repeat a total of 5 times.

Measurements under microplate reader

  1. Mix two bottles of TMB reagent (Solution A & B) by a volume ratio of 1:1, vortex if needed.
  2. Add 50μL of TMB mixture to each well
  3. Incubation in dark for 15min at 37°C
  4. Reaction stopped by the addition of stopping buffer(2M of H2SO4)
  5. Move the solution into 96-well plate, 75μL per well.
  6. Fluorescence is measured by microplate reader at absorbance=450nm

We employed this method testing the binding affinity of HR2 aptamer(Figure 1).

Figure 1 A qualitative test for the binding affinity of HR2 aptamer using MB-ELONA.

As can be observed in the result, BSA-coated MB showed OD450 values of roughly 0.2 in both aptamer concentrations, proving that our aptamer did not bind to BSA protein; whereas HER2-coated MB showed an increase in OD450 value when aptamer concentration increased by 0.1 when aptamer concentration increased from 0.0 to 1.0 μm. This provided evidence that our aptamer does have some degree of affinity for HER2 protein. We also performed MB-ELONA with no protein-coated, this gave us insight into possible reasons for the lack of difference between the control and experiment groups. We also performed MB-ELONA with no-protein conjugated. This proves the validity of MB-ELONA, as aptamers bind non-specifically to MB, which means that proteins were actually coated onto MB.

ELISA-Kit Modified ELONA

We bought ELISA kits and modified them to suit our needs. We designed our experiment based on a sandwich ELISA kit which is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of human HER2 in serum, plasma, and cell culture supernatants. Our assay employs a well-plate with an antibody specific for human HER2 receptors coated on a 48/96-well plate. Recombinant Human HER2 standard is pipetted into the wells and is bound by the immobilized antibody. The wells are washed and this time we used FAM-labeled aptamers to reduce the rounds of incubation needed to increase the efficiency of the experiment.

    Materials

  • ELISA Kit

    • 96-well plate with immobilized antibody

  • Aptamer Modification: Biotin Labelling/FAM

  • HER2 ECD Protein from ELISA Kit

  • CBS Buffer

  • Blocking Reagent

  • PBST (1*PBS + 0.1% Tween 20)

  • Binding Buffer Solution (BBS) (MgCl2 + PBS)

  • (TMB Reagent)

  • (Streptavidin-HRP)

  • (Sulphuric Acid at 2M)

Coating HER2 on 96-well plate with recombinant protein

Coating Procedure:

  1. Prepare dilute HER2 ECD protein solution at final concentration of 4 ng/mL with sample dilutant.
  2. Add 50 μL/well for 96-well plate wells.
  3. Incubate for 1 hour at RT

Binding of Aptamer with HER2 ECD

  1. Dilute Biotin-labelled or FAM aptamer to desired concentration μM by BBS buffer; place the aptamer on 95°C for 5 min, then immediately on ice for 15 min.

  2. Add 50 μL of diluted aptamer into each well.

  3. Incubation at 37°C for 1h, wash 6 times with PBST, 3 min each time

      Notes: If FAM-Aptamer is used, then it can be measured after this step, using fluorescence intensity.

  4. Add 100uL of 1/2000 diluted Streptavidin-HRP

  5. Incubation at 37 ℃ for 1 hour, wash 6 times with PBST, 3 min each time

Measurements Under Microplate Reader

  1. Mix two bottles of TMB reagent (Solution A & B) by a volume ratio of 1:1, vortex if needed
  2. Add 100 μl of TMB mixture to each well
  3. Incubation in dark for 15min at 37°C
  4. Reaction stopped by the addition of stopping buffer(2M of H2SO4) 25 μL
  5. Fluorescence is measured by microplate reader at absorbance=450nm

Before any experiment, we evaluated the sensitivity of the Kit by testing with its standard sample provided (Figure 2).

Figure 2 Standard test of the ELISA Kit.

We chose to coat 4 ng/mL of protein onto the provided well-plate as shown in the result it showed a significant level of value, meaning it contains enough HER2 to bind with aptamer.

Our design was successful and we were able to obtain a qualitative result to prove the specificity of our aptamer, followed by a full quantitative evaluation of HR2 aptamer. (See more in Results)

Figure 3 (left) Qualitative result of ELONA. (right) Quantitative evaluation of HR2 aptamer

Conclusion

The main measurements involved in our Oncokiller project are MB ELONA and ELISA-Kit Modified ELONA. We use these methods to test the binding affinity of aptamers against HER2 protein qualitatively and quantitatively. This experimental period is of trying, failing, and trying again to break through the barrier of negative results and obscure curves. We kept trying different variations of ELONA methods，such as MB-ELONA, Traditional ELONA, HER2 ELISA-Kit method, and ELISA-Kit modified ELONA. Fortunately, we peep into the potential for development, when we introduce the HER2 ELISA-Kit to our experimental design. We took this opportunity to continually enhance our design, optimize the experimental process and improve detection methods. The finally developed measurement- ELISA-Kit modified ELONA, not only does this method improve detection sensitivity and accuracy by a large margin, but also raises detection efficiency by reducing the experimental period by half.

We set up strict controls during the experiment and through multiple trials, we were able to obtain the binding curve of HR2 aptamer against HER2 protein. This proves the validity and the repeatability of our protocol.