Difference between revisions of "Team:GreatBay SCIE/Measurement"
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<h1>ELISA-Kit Modified ELONA</h1> | <h1>ELISA-Kit Modified ELONA</h1> | ||
<p>We bought ELISA kits and modified them to suit our needs. We designed our experiment based on a sandwich ELISA kit which is an <em>in vitro</em> 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.</p> | <p>We bought ELISA kits and modified them to suit our needs. We designed our experiment based on a sandwich ELISA kit which is an <em>in vitro</em> 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.</p> | ||
| − | <p>Before any experiment, we evaluated the sensitivity of the Kit by testing with its standard sample provided (Figure | + | <p>Before any experiment, we evaluated the sensitivity of the Kit by testing with its standard sample provided (Figure 2).</p> |
<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2021/6/6e/T--GreatBay_SCIE--Results_Figure_1_Standard_Test.png" width="80%"> | <img src="https://static.igem.org/mediawiki/2021/6/6e/T--GreatBay_SCIE--Results_Figure_1_Standard_Test.png" width="80%"> | ||
| − | <div class="image_text">Figure | + | <div class="image_text">Figure 2 Standard test of the ELISA Kit.</div> |
</center> | </center> | ||
<p>We chose to coat 4 ng/mL of protein onto the provided well-plate as shown in result it showed a significant level of value, meaning it contain enough HER2 to bind with aptamer.</p> | <p>We chose to coat 4 ng/mL of protein onto the provided well-plate as shown in result it showed a significant level of value, meaning it contain enough HER2 to bind with aptamer.</p> | ||
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<center> | <center> | ||
<img src="https://static.igem.org/mediawiki/2021/4/4a/T--GreatBay_SCIE--ELONA_Qualitative.png" width="50%"><img src="https://static.igem.org/mediawiki/2021/6/6a/T--GreatBay_SCIE--ELONA_Quantitative.png" width="50%"> | <img src="https://static.igem.org/mediawiki/2021/4/4a/T--GreatBay_SCIE--ELONA_Qualitative.png" width="50%"><img src="https://static.igem.org/mediawiki/2021/6/6a/T--GreatBay_SCIE--ELONA_Quantitative.png" width="50%"> | ||
| − | <div class="image_text">Figure | + | <div class="image_text">Figure 3 (left) Qualitative result of ELONA. (right) Quantative evaluation of HR2 aptamer</div> |
</center> | </center> | ||
Revision as of 12:17, 19 October 2021
Measurements
MB ELONA
See our protocol summary for more information about the experiment.
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.
We employed this method testing the binding affinity of HR2 aptamer(Figure 1).
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 on 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.
Before any experiment, we evaluated the sensitivity of the Kit by testing with its standard sample provided (Figure 2).
We chose to coat 4 ng/mL of protein onto the provided well-plate as shown in result it showed a significant level of value, meaning it contain enough HER2 to bind with aptamer.
Our design were successful and we were able to obtain a qualitative result to prove the specificity of our aptamer, followed by a full quantitative evalutation of HR2 aptamer. (See more in Result)
