For pathogen detection, the objective was to use the following experimental procedure:
First, infected mosquitoes are grinded in order to expose pathogen proteins. Then, mosquitoes proteins and pathogen proteins of the sample are bind to horse-radish peroxidase (HRP). The conjugated proteins are deposited on are the patch precoated with specific antibodies. The patch is then washed, pathogen proteins are retained by antibodies, and peroxidase activity is revealed using a specific substrat.
The principle of that method had to be checked before running experiment. In particular, specificity and sensitivity had to be evaluated. For those tests, we used purified viral proteins (E-protein from Yellow fever virus or E2 protein from chikungunya virus) and specific antibodies (4G2 for E-protein of Yellow fever virus and 3E4 antibody for E2 protein from chikungunya virus).
We first checked that we were able to bind viral protein with HRP, then that we could detect viral protein on a membrane, either purified or diluted in a Bovine Serum Albumine (BSA) solution in order to mimic mosquitoes proteins. Next step was to test the method when viral proteins were diluted with mosquitoes proteins. Finally, we tested the patches ability to detect viral pathogens from infected mosquitoes. For this last experiment, all steps involving infectious materials were performed by one of the coaches in a BSL3.
General protocol:
Materials:
• PVDF or nitrocellulose membrane
• YFV E protein, CHIKV E2 protein
• 4G2 antibody
• Secondary antibody (anti-mouse alexafluor 488)
• BSA
• PBS
• Tween
• Milk 5% in PBS-Tween
• EZ-link kit
• Rocker agitator
Method:
Sample preparation:
The sample is tagged using EZ-Link kit.
1. Add 500mL of ultrapure water to the dry-blend Phosphate Buffered Saline (PBS).
2. Prepare 1mg of antibody in 1.0ml of PBS(+/-BSA1%).
3. Reconstitute 1mg of lyophilized EZ-Link Plus Activated Peroxidase with 100 µl of ultrapure water and add it to the antibody solution or add the protein sample directly to the lyophilized activated.
4. In a fume hood, immediately add 10 µl of Sodium Cyanoborohydride to the reaction and incubate for 1 hour at room temperature.
5. Add 20 µl of Quenching Buffer and react at room temperature for 15 minutes.
→ The conjugate can be store at 4°C for up to 4 weeks.
Membrane:
1.
Coating: put 1 µl of a non-diluted antibody on the membrane, let the membrane dry for 5 minutes at room temperature.
2.
Saturation:
• Put the membrane in PBS-Tween 0,5%-milk 5% on a rocker, 1 hour at room temperature.
• Replace the washing solution with a fresh one.
3.
Binding: Add the sample in the PBS-Tween 0,5%-milk 5% solution at the appropriate dilution, incubate overnight at 4°C on a rocker.
4.
Washing: Wash the membrane 3 times for 5 minutes on a rocker at room temperature.
5.
Revelation: Reveal the membrane using approximately 1ml of Pierce ECL Western Blotting substrate.
August 4th, 2016 :
EZ-link binding test: Proof of concept of the technic
Aim:To test the technic, we used our protocol and checked that we were able to detect pure viral protein bind to EZ-link. We used the previous protocol, on the membrane, we coated 4G2, BSA was used as negative control. On the membranes, we incubate the membrane with the following solution. Negative control are membrane A to D, to validate the experiment, membrane E has to be positive.
• A: YFV E-protein + HRP EZ-link
• B: YFV E-protein only
• C: PBS + HRP EZ-link
• D: BSA+ HRP EZ-link
• E: YFV E-protein + HRP EZ-link
Results:
Conclusion: The principe of the technic is validated.
Aim: Sensitivity of the detection.
To evaluate the sensitivity, we proceed as described before, and we used YFV E protein pure or serially diluted into PBS + BSA 1% to mimic the presence of mosquito proteins in the sample. 3 dilutions were used : 1mg/ml, 10-4mg/ml, 10-8mg/ml.
Results: No signal was visible.
Aim: Sensitivity of the detection.
The previous experiment was repeated, we made new reagents and used nitrocellulose membrane in addition to PVDF membrane.
Results: No signal was visible.
Aim: Sensitivity of the detection
The previous experiment was repeated, we used a new antibody (anti-chik 3E4) together with CHIKV E2 envelope protein.
Results:
Aim: Sensitivity of the detection with mosquito protein.
100 mosquitoes were grinded and CHIKV E2 envelope proteins were diluted into in order to have approximately the same amount of viral proein we would find into one infected mosquito alone and in one infected mosquito together with 100 non-infected mosquitoes in order to mimic the fact that not all mosquitoes trapped will be infected with a pathogen.
Results:
Aim: Proof of concept.
In this experiment, we used 8 prototype patches to check their ability to detect pathogens in mosquitoes.
4 patches were used to repeat the previous experiments in real conditions. The 4 patches remaining were used to test infected mosquitoes. Aedes aegypti were experimentally infected with the vaccinal strain of Yellow fever virus. 14 days after infection, they were grinded in 1,5ml tubes. Tubes were centrifuged and supernatant were inactivated by placing the tubes under a UV lamp for 30 minutes. All steps using infectious materials were performed by a coach in a BSL3. .
Samples were then tested on the patch using the same protocol as decribed previously with the patch as a solid support for antibody (in remplacement of membranes).
Results:
Patches are able to detect high amount of viral proteins, the sensitivity seems to be improved for detecting pathogens at a lower level.
