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.
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.
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.
You are officially The Best.
Curious if anyone ended up running this process, and, if so, what your results were?
Agree with John, thank you so much!