Team:GreatBay SCIE/Measurement

ShroomSweeper GreatBay_SCIE



The main measurements involved in our ShroomSweeper project are the ELONA[1] (Enzyme-Linked OligoNucleotide Assay), the ELISA[2] (Enzyme-Linked ImmunoSorbent Assay), and the competitive binding experiments[3]. They are used by us to quantitatively analyze the binding of our aptamer and scFv ligands to the target molecules.

ELONA protocol

We used the following experimental protocol for ELONA experiments:

1. Coating of target on 96-well plate

  1. Prepare Coating Buffer Solution (pH9.6 NaHCO3-NaCO3) (CBS).
  2. Prepare target molecule solutions with mass concentration 40 ng/mL using CBS.
  3. Add 100 µL of target molecule solution to the designated wells for experiment.
  4. Put inside 4 degrees Celsius refrigerator overnight (>8 hours).
  5. Drain the liquid.
  6. Add 100 µL PBST to each well used for experiment.
  7. Put on vortex mixer for 3 minutes.
  8. Drain the liquid in each well.
  9. Repeat steps 6-8 for 5 times, for 6 times in total.
  10. Add 100µl blocking solution (50 mg/mL solution of skimmed milk powder dissolved in PBST) to each well.
  11. Block for 2 hours.
  12. Drain the liquid.
  13. Repeat steps 6-8 for 6 times.

2. Conjugation

  1. Prepare 800nM solution of biotin-modified aptamers using PBS.
  2. Add 100 µL of aptamer solution to each well.
  3. Incubate at 37 degrees Celsius for 2 hours.
  4. Drain the liquid.
  5. Repeat steps 6-8 in coating(add PBST, vortex and drain the liquid)for 6 times.
  6. Prepare 1/2000 diluted streptavidin-HRP solution.
  7. Add 100 µL of streptavidin-HRP solution to each well.
  8. Incubate at 37 degrees Celsius for 2 hours.
  9. Drain the liquid.
  10. Repeat steps 6-8 in coating(add PBST, vortex and drain the liquid)for 6 times.

3. Measurement and Analysis

  1. Mix two components of TMB reagent by a volume ratio of 1:1, and vortex to mix thoroughly.
  2. Add 100 µL of this mixture to each well.
  3. Incubate at 37 degrees Celsius for 15 minutes without light.
  4. Add 100µL of 2M H2SO4 solution to each well to stop the reaction.
  5. Using a spectrometer, measure the absorbance of light with wavelength 450 nanometers of the liquid in each well.

ELISA protocol

We used the following experimental protocol for ELISA experiments:

1. Coating of target on 96-well plate

  1. Prepare Coating Buffer Solution (pH9.6 NaHCO3-NaCO3) (CBS).
  2. Dilute 5 µL 20µg/mL amatoxin solution to 100 µL using CBS
  3. Add 100 µL of target molecule solution to the designated wells for experiment.
  4. Put inside 4 degrees Celsius refrigerator overnight (>8 hours).
  5. Drain the liquid.
  6. Add 100 µL PBST to each well used for experiment.
  7. Put on vortex mixer for 3 minutes.
  8. Drain the liquid in each well.
  9. Repeat steps 6-8 for 5 times, for 6 times in total.
  10. Add 100µl blocking solution (50 mg/mL solution of skimmed milk powder dissolved in PBST) to each well.
  11. Block for 2 hours.
  12. Drain the liquid.
  13. Repeat steps 6-8 (add PBST, vortex and drain the liquid)for 6 times.

2. Conjugation

  1. Add 50-100 µL antibody solution to each well.
  2. Incubate at 37 degrees Celsius for 1 hour, without light.
  3. Drain the liquid.
  4. Repeat steps 6-8 in coating(add PBST, vortex and drain the liquid)for 6 times.
  5. Prepare anti 6*Histag-HRP solution solution: 1:5000 dissolve in PBST. All operation performed on ice.
  6. Add 100 µL of anti 6*Histag-HRP solution to each well.
  7. Incubate at 37 degrees Celsius for 1 hour, without light.
  8. Drain the liquid.
  9. Repeat steps 6-8 in coating(add PBST, vortex and drain the liquid)for 6 times.

3. Measurement and Analysis

  1. Mix two components of TMB reagent by a volume ratio of 1:1, and vortex to mix thoroughly.
  2. Add 100 µL of this mixture to each well.
  3. Incubate at 37 degrees Celsius for 30 minutes without light.
  4. Add 50µL of 2M H2SO4 solution to each well to stop the reaction.
  5. Using a spectrometer, measure the absorbance of light with wavelength 450 nanometers of the liquid in each well.

Measurement results

The quantitative result of either ELONA or ELISA is given as a number. This represents the relative amount of 450 nanometer-wavelength light absorbed by the resulting solution. Control groups like BSA/water and groups without aptamer are set up. To improve coating, we conjugate our amanitin to BSA, which is a larger protein and is easier to be coated onto the 96-well plate, as suggested by Dr.Bever.

During the conjugation step, an amount of ligand is added to the wells coated with the target molecules. After some time of incubation, the ligand is given the chance to bind with the targets. Any unbound ligand is washed away. The remaining bound ligand on the walls of the well is conjugated with HRP, which then gives a yellow color under reaction with the TMB reagent and sulfuric acid. It was found that there is a positive relationship between the amount of ligand that is bound to the target molecule and the "yellowness" of the resulting solution, in which this "yellowness" could be quantitatively measured by a spectrometer measuring OD450 absorbance.

In simplified terms, the higher the absorbance in the ELONA/ELISA results, the better the ligand binds to the target.


ELONA experiments for previously reported H06 aptamer[1] and Best 1 aptamer[4] for alpha-amanitin

We carried out ELONA verification of binding of an alpha-amanitin aptamer referred to as H06, reported in previous researches, and plot a graph to present the absorbance.

Fig.1 Results of the ELONA on H06.

After this success, we have faced several failures in ELONA. In the interview with Dr. Candance Bever, she tells us that amanitin is so small that it is hard to immobilize, and suggest us to conjugate amanitin with BSA, a larger protein, which is easier to coat on. This is a very useful notice in experiments where immobilization of amanitin is needed.

Fig.2 Results of the ELONA on Best1.



We then conjugate α-amanitin to BSA, with the help from Dr.Bever, and conduct ELONA on α-amanitin with Best 1, another reported aptamer of α-amanitin. As the graph clearly points out, the absorbance in the group of the α-amanitin-BSA conjugate is much higher than BSA or water, even higher than α-amanitin itself. The power of conjugation is therefore verified, and we decide to use the same tactic to experiment on our own aptamer.


ELONA experiments of SELEX_selected aptamers for beta-amanitin

We then carried out ELONA verification of binding of the beta-amanitin aptamers 1 and 2 with beta-amanitin, beta-amanitin-BSA conjugate, and pure BSA negative control.

Plotting the data into Box-and-Whisker graphs, we obtain the following for aptamers 1 and 2:

It can be seen that both of the groups of β-amanitin-BSA conjugate have much higher absorbance than other groups, and the affinity is therefore proved.


ELISA experiments for scFv for alpha-amanitin

For the scFv for α-amanitin[2] we produced using Escherichia coli, three groups of ELISA experiments were carried out to verify its binding ability towards alpha-amanitin. It is worth mentioning that the second group, ic-ELISA, refers to “Indirect Competitive ELISA”, in which free alpha-amanitin were added during the binding step of the scFv and the immobilized toxin on the walls of the well, in order to achieve the effect of competitive binding. The results are listed in the following:

The OD450 absorbance for normal ELISA was the highest, followed by that of ic-ELISA, and that of the negative control group remains the lowest. This demonstrates the specificity of scFv towards alpha-amatoxin, and the possibility of a competitive test.

Measurement in Competitive Test

The design of our hardware involves a competitive test, so we need to work out the limit of detection of aptamer in a simulation of competitive tests. Thus, we conduct competitive tests between the molecules conjugated to magnetic beads and free molecules in solution.

Steps of Competitive Test

Prepare B&W Buffer: 10mM phosphate, 138mM NaCl, 2.7mM KCl, 2.5mM MgCl2, PH7.4

  1. Several groups of aptamers of different concentrations (diluted by PBS) are mixed with different concentrations (diluted by PBS) of free molecules.
  2. The mixture then are added to molecule(amanitin/thrombin) functioned magnetic beads, incubating for 40 minutes.
  3. Wash(add B&W buffer to the mixuture, put the EP tube on the magnetic stand, extract and discard the supernatant) for 6 times.
  4. After washing, the aptamers that bind to the free molecules are washed away, and the rest is bound to the immobilised molecules. Add water to the magnetic beads and elute the aptamers down under high temperature.
  5. Extract the supernatant that contains the aptamer and amplify it through PCR.
  6. Conduct gel electrophoresis and check the bands.

Measurement Results

The result is obtained through PCR and gel electrophoresis, and measured through the light intensity of the fluorescence. To demonstrate the measurement of the Competitive Test, we have an example of a competitive test on thrombin here.

Here we attempt to further decrease the limit of detection by reducing the aptamer concentration, based on our conclusion of model 2, with concentration gradients of both thrombin and its aptamer, and with non-competitive tests and a group of high as control. The principle is verified by a significant decrease of LOD, characterized by the difference of light intensity of the electrophoresis band at 100 times lower concentration (13.5uM) compared to the original concentration (1.35mM).

Reference

  1. Han Q, Xia X, Jing L, et al. Selection and characterization of DNA aptamer specially targeting α-amanitin in wild mushrooms. SDRP J Food Sci Technol. 2018;3(6):497-508. doi:10.25177/JFST.3.6.2
  2. Zhang X, He K, Zhao R, Feng T, Wei D. Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors. Food Anal Methods. 2016. doi:10.1007/s12161-016-0509-3
  3. Jauset-Rubio M, Svobodová M, Mairal T, et al. Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers. Anal Chem. 2016;88(21):10701-10709. doi:10.1021/acs.analchem.6b03256
  4. Muszyńska K, Ostrowska D, Bartnicki F, et al. Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides. Acta Biochim Pol. 2017;64(3):401-406. doi:10.18388/abp.2017_1615