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Hi,
I wrote down this manual for anyone to follow - https://drive.google.com/file/d/1qtfyE42ECZAqSFpqGR9JawX3YoVAvVSg/view?usp=sharing
As it is a modified protocol I'm not sure It will work (especially when we do not have any positive and negative baseline).
In short:
Antigen (peptide) binding to the plates:
- Prepare peptide solutions in PBS buffer in 1.5 mL tubes. The final concentration of the peptides should be around 10-20 µg/mL
- Prepare high-binding 96-well plates and a piece of tin foil for cover. Add a 100 µL of peptide solution to each experimental well. Cover the plate and incubate at 4oC over night (16h).
- Take the plate from the fridge and discard peptide solution
- Wash the plate by adding wash buffer (approx. 200 µL per well).
Blocking:
- Add approx. 200 µL of blocking solution to each well. Incubate for 2h at room temperature and discard.
- Wash the plate 3 times as described above.
Addition of the primary antibodies (from nasal swabs):
- Prepare the nasal swab/wash samples. Collected swabs should be stored in PBS. Nasal wash can be performed using PBS. The samples can be applied to the wells without dilution, but I would suggest further dilution of the samples using antibody buffer.
- Add prepared nasal samples – 100 µL/well. Incubate at 4oC overnight.
- Discard samples and wash the plate 4 times as described above.
Addition of secondary antibodies conjugated with HRP:
- Prepare the solution of secondary antibodies (1:2500) in antibody buffer.
- Add secondary antibodies to the plate – 100 µL/well. Incubate for 90 min. at room temperature.
- Discard secondary antibodies and wash 5 times as described above.
- Add 100 µL of TMB to each well. The reaction should turn the solution blue. The time of the incubation can vary from 1 min. to 30 minutes.
- If you want you can stop the reaction by adding 50 µL of 0.5 M H2SO4 – this will turn solution yellow.
I guess anything with a signal stronger then control can be considered positive signal. Usually for my experiments I'm using the cutoff of "three times value of the blank or more (after subtraction of the proper negative control)". If you do not have negative control you can only use blank as a point of reference.
I think that you can perform ELISA using unpurified nasal wash or diluted mucus. I often perform ELISA using sera/full blood or unpurified cell lysates. I would do a nasal or throat swab and put it in phosphate buffer (or PBS). You can further dilute samples if the viscosity is too high. ELISA is very sensitive and my guess is, you should be able to see positive signal without plate reader. It very much depends on both antigen used in ELISA and antibody concentration. For peptides such as yours (without tags) I use concentration of about 10-20 ug/mL, sera diluted 1:1000-1:10000 and higher and still get good, clear signal. Of course you don't want to dilute your nasal samples so much, but undiluted, 1:5 or 1:20 could give nice reading, hopefully without any background. EDIT: I know it may sound complicated, but I just wanted you to know that checking immune response against antigen used (in this case peptides) is a possibility.
This may be true, I'm talking about ELISA test. In theory you only need high binding plates, secondary antibodies and some kind of detection (TMB + H2SO4 give colorimetric effect and you can try and skip plate reader). I understand that it can be quite complicated to perform, but I am wondering how you plan to chceck the immune response against Orf1 and Orf 8. You could check blood against Spike and nucleocapside (you need to check if antigen test is using full length Spike and Nuc), but due to the use of peptides, lenghth of the peptides and expected low IgG titer, detection (as you said) can get tricky. But congrats anyway.
Hi,
It's quite interesting what you are doing. Have you thought about testing immune response yourself using peptides from the vaccine? Of course you can only run humoral responses, but it would give you far more accurate results then any commercially available test. You could bind peptides to the high-binding plates, use your mucosa samples and detect with universal anti-human IgM+IgG+IgG antibodies conjugated with HRP.