Continuation on Quantifying SiteClick Membrane Efficacy
The purpose of this experiment was to quantify the effectiveness of the kit membrane at separating free antibody and qdots from qdot-bound antibodies. The manual advertises an approximate 80% yield for this step and no purity reading.
It was originally planned to directly measure the purity based on the amount of free antibody in the pre-filtration vs post-filtration solutions, however the extremely low concentration of free antibody in both stages made this measurement impossible. Instead a more indirect method was chosen which is to measure the total amount of antibody before and after the filtration. This method works based on the assumption that in all trials of the experiment, the initial concentration of free antibody is the same and that the purification process is the same for each stage so that the measurement can be made as direct as possible.
The purity can be calculated using this new method based pre-existing measurements and is the simplest procedural method of all previous methods. The absorbance of 280-250 of the pre-filtered solution is measured and the volume is recorded, then the solution goes through serial dilutions and filtrations to obtain a purified sample, and the absorbance of 280-250 is measured along with the final volume. The pre and post filtration amounts of antibody are compared and it is assumed that all post-filtration measured antibody is bound. This experiment will be repeated to create a reliable post-filtration antibody amount.
This experiment will then be performed on the nanomembranes (with exactly the same preparation procedures to ensure the same initial amount of free antibody) in order to compare the two methods’ post-filtration antibody amounts.
Results
The results of this experiment are that the mass of antibody lost was approximately 42.5% with as standard deviation of 5.43% when the same sample was measured twice.
It was also found that the amount of qdots did not decrease, meaning that either all qdots were conjugated or the membrane failed to filter out unconjugated qdots.
Drawbacks
The primary drawbacks of this method are:
- It is not a direct measurement of the efficiency of the membranes, it can only be used to compare efficiencies
- The initial amounts of free antibody must be the same in all measurements, meaning that all preparations must be exactly identical.
- The initial concentration of qdot provided by the kit is unknown, so actual concentration values of qdots cannot be calculated.
Future Plans
1) This method of measuring the efficiency of the membranes will be used on two other kits to verify the results of the provided kit membrane.
2) The design of the desired nanoporous chip must also be decided upon by doing centrifugation separations with 40nm pore chips with one loaded with 5 nm gold, and one with 1 um yellow beads, ensuring that the 5nm gold go through while 1um yellow is held back. Also attempt to get the spin times and flow through rates as similar to that of the kit as possible.
3) This method will be used to test the nanoporous chips in that the kit preparation of the solution will be identical to the tests with the provided membrane such that the number of initial conjugated qdots can be assumed to be the same.
Future binding test
The next stage in determining the efficacy of these membranes is quantifying how the membrane effects the binding power of the antibody to a surface. This testing will be done in conjunction with Kilean’s current project in testing the adherence of exisomes to a surface. The exact numbers and method are TBD.
