Our wet lab team completed a literature review to find successful protocols for the design of our diagnostic test and the expression of our antibody sequences in E. coli SHuffle. Due to COVID-19 restrictions, we primarily spent our laboratory time working on the construction of our lateral flow assay and the obtainment of values for our modeling and hardware sub-teams. Click more to see the protocols we used and hypothesized for the completion of this project.
Collection of Menstrual Effluent
Collection Method and Transport (Nayyar et al., 2020)
Patients will be instructed to wear the menstrual cup for 4 to 8 hours on the day of their heaviest flow. The sample will then be stored in a test tube to be transported to the clinic or laboratory either on ice or refrigerated at 4℃ immediately after collection.
1. Collection will occur on the second or third day of the patient’s menstrual cycle.
2. Samples will be centrifuged at 1,500 rpm for 30 minutes.
3. Supernatants will be collected using a pipette.
ME can contain tissue and clots. The amount of anticoagulant, heparin, may have to be altered to account for this difference between menstrual and peripheral blood. If there is not sufficient anticoagulant in the tube, the excess clotting and thick viscosity of the blood should be visually apparent and require more steps of serial processing and centrifugation. If the problem persists after addition of more heparinization, other methods have cooled the blood samples and physically removed the clots from the blood samples prior to centrifugation. However, the simple use of a heparinized tube for this processing step should minimize the risk of poor serum separation outcomes. Additionally, clots can form in the serum after centrifugation. These clots should be removed by removing the tube stopper and then wrapping the fibrin clot around a wooden stick applicator. Alternatively, the sample can be re-spun. To prevent serum clot formation, the sample should sit in the tube for at least 30 minutes prior to centrifugation.
1. 500 uL of menstrual effluent (ME) will be plated per T-75 flask in growth media.
2. Samples will be incubated at 37℃/5% CO2 for 24 hours.
3. Samples will be aspirated and washed with 1x PBS and the growth media will be replaced.
4. Samples will be monitored and the growth media will be replaced every 3-4 days for the course of the experiment.
5. Samples will be passaged 1:6 after trypsinization.
6. Collection of menstrual effluent-derived stromal fibroblast cells (ME-SFCs).
7. 1.5 x 104 ME-SFCs will be plated in 96-well plates in 200 uL growth media and grown until confluent.
8. Samples will be incubated at 37℃/5% CO2 in decidualization media and induced using either 0.5 mM 8-bromoadenosine 3’, 5’ cyclic monophosphate sodium salt or 1x PBS (as a vehicle) for 24 hours.
9. After a step of brief centrifugation, collect the supernatants of the cultures.
DMEM with 10% FBS, 1% penicillin/streptomycin, 1% glutamine, Normocin (1:500)
DMEM with 2% FBS, 1% penicillin/streptomycin, 1% glutamine, Normocin (1:500)
Incubate IGFBP-1 for 2 hours at 22 degrees Celsius in 0.3 ml of 0.05 M sodium phosphate buffer and 0.25 % bovine albumin, pH 6.5.
This protocol has been optimized by the labs pursuing this research. Troubleshooting this protocol will likely consist of collaboration with our contacts Dr. Peter Gregerson and Dr. Christine Metz. We can measure the amount of IGFBP-1 present using an ELISA assay prior to testing it on our test strip to verify that sufficient amounts are present and consistent with those previously reported in literature. Verification of cell type can be performed using flow cytometry to negate the chances of contamination by other cells present in the menstrual effluent. If we do not produce optimal results (a high yield of IGFBP-1) using these methods, we will run our assay at various periods of incubation with IGF-1 to see which produces the best results. This can be done by measuring the binding of IGF-1 and IGFBP-1. This could be done by using His-tagged IGFBP-1 and incubating it with an equal amount of IGF-1. His tag purification can then be used to isolate the His-tagged IGFBP-1 and the collected protein can be run on a SDS-PAGE gel. Two bands should be present at the molecular weights of IGFBP-1 and IGF-1 and the intensities can be compared to see the proportion of bound versus unbound IGF-1. For example, if the bands were equal in intensity that would indicate almost all of the IGF-1 was bound to IGFBP-1. If the band for IGF-1 is lighter than that of IGFBP-1, that would indicate that not all IGFBP-1 was able to bind IGF-1. In this case, the intensity of the bands could be compared to solve for the relative proportions of IGFBP-1 to IGF-1 to see how efficient binding is for these molecules.
Use of Gold Nanoparticles (GNPs)
A gold nanoparticle conjugation kit was purchased commercially from Abcam.
Troubleshooting a poor yield for gold nanoparticle attachment would include independently altering the incubation times and buffer compositions to produce optimal yield. The yield of labeled antibodies would be measured by isolating our antibodies using standard purification methods such as affinity-based chromatography and measuring the intensity of color in the isolated fraction or sample suspected to contain the antibody. High intensity would indicate that the gold nanoparticles have bound well to our antibody, while low intensity would indicate a lack of binding. Intensity would be measured using pre existing sensors that measure the optical properties of solutions. This could be compared to intensity values correlated with gold nanoparticle concentration. Prior to our experiment, we could create known dilutions of gold nanoparticles and measure their intensity. Once the intensity was correlated with concentration, we can compare the intensity with our expected concentration of antibodies. If we were unable to troubleshoot this protocol, we may also look at other existing protocols for attachment of GNPs to antibodies that use different methods. Color intensity may also be affected by the properties of the gold nanoparticles. We will use transmission electron microscopy to ensure our gold nanoparticles are uniformly spherical and 40 nm.
Lateral Flow Assay Set Up
1. Prepare test strips using a width of 5 mm and length of 10 cm for each material.
2. Deposit (0.2 x 10-4 ug) of antibodies on a 5 mm2 section of the test strip, approximately 20 mm from the start of the strip.
3. Deposit (0.16 x 10-4 ug) of antibodies on a 5 mm2 section of the test strip, approximately 25 mm from the start of the strip.
4. Deposit 16 uL of labeled antibodies onto the conjugate pad.
5. Allow the test strips to dry at 40 degrees Celsius.
6. Soak the nitrocellulose membrane in a blocking buffer and shake the membrane at room temperature for 30 minutes.
7. Allow the membranes to dry at 40 degrees Celsius.
8. Use the adhesive on the plastic backing to construct the test strip. Place the conjugate pad at the start of the test strip and overlap the nitrocellulose membrane by 2 mm. Place the sample pad on top of the conjugate pad, following the diagram below. Place the absorbent pad so it overlaps the opposite end of the test strip by 2 mm.
It can be difficult to produce a successful lateral flow assay without several rounds of guessing and checking the materials and concentrations. Location of the test line and reagent concentrations were optimized using modeling software to address this possible error in setup of the assay itself. To test the success of our design, we will run our lateral flow assay with known concentrations of our target molecule and read the colorimetric signal at the test line to see if it properly correlates with our target concentration. If the signal does not correlate with the concentration, we will alter the design of the lateral flow assay with a “guess-and-check” approach as outlined in the guide “Rapid Lateral Flow Test Strips: Considerations for Product Development” from MerckMillipore. Possible areas for alteration include the selection of different membranes, such as altering the pore size of the nitrocellulose membrane, and the concentrations of reagents used. Any changes would be performed independently and compared to the original design to determine their effect on signal output.
Gold Enhancement Solution
Immediately before application to test strip, mix equal volumes of solutions of hydroxylamine (2 mM in H2O) and 1% HAuCl4 in H2O.
1. Apply 20 uL of enhancement solution to wet test strips in the test zone area.
2. Hold in place for three minutes.
3. Wash with water.
If the amplification of our signal by gold enhancement solution is not sufficient for our desired detection threshold, the best approach would be to alter the ratio or concentration of the reactants individually to see what produces the optimal results. This could be done by measuring the change in intensity that is observed by altering the ratio of solution components. A known concentration of labeled antibodies could be placed on the strip (ideally close to our intended biomarker concentration) and then saturated with solution. The intensity could then be read once the strip dried to inform our approach what the best concentration of reactants is to produce the highest intensity level.
Additionally, we could vary the time the test strip spends in the enhancement solution to determine the optimal application time. Using transmission electron microscopy (TEM), we will visualize and characterize the size of the gold nanoparticles before and after the addition of enhancement solution to confirm uniformity and enlargement.
Protocols for Plasmid Transfection and Antibody Isolation
E. coli SHuffle Transfection (New England BioLabs)
1. Thaw a tube of SHuffle Competent E. coli cells on ice for 10 minutes.
2. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 30°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 30°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 30°C. Alternatively, incubate at 25°C for 48 hours.
If transformation is unsuccessful, we may want to try electroporation instead of heat shock. If we have a low yield of E.coli transformants, then our protein may be toxic to the cell and we may want to find a lower copy number plasmid or lower the incubation temperature. Media may also be recreated with different concentrations of the selected antibiotic marker, to ensure the concentration is not too high and kills off transformed colonies or too low and allows for contamination. If we have a high yield of E.coli without the transforming DNA, then we could test if untransformed E.coli survives on the selection antibiotic. If they do, a different antibiotic could be used or the cells with the recA mutation should be used to prevent the vector plasmid from recombining with the chromosomal DNA.