All of Anna Czarnota's Comments + Replies

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:

  1. Prepare peptide solutions in PBS buffer in 1.5 mL tubes. The final concentration of the peptides should be around 10-20 µg/mL 
  2. Prepare high-binding 96-well plates and a piece of tin foil for cover. Add a 100 µL of peptide sol
... (read more)
5jacobjacob3y
Curious if anyone ended up running this process, and, if so, what your results were? 
1kjz3y
Agree with John, thank you so much!
7johnswentworth3y
You are officially The Best. 

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 ... (read more)

5johnswentworth3y
This was a very useful thread. Thankyou and strong upvote.
2kjz3y
Is this something that can be done at home with readily available and affordable equipment? If so, would you be willing to share more details of how someone might get started? I think a lot of readers would be interested in hearing more about this - it could even be its own post.
2ChristianKl3y
Thanks for explaning it. Maybe someone here will put it into practice.

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.

2ChristianKl3y
Back in university I did a ELISA test. It's a bit fuzzy in my memory but I think we did a lot to purify the solution we were working with before we did ELISA. Do you think the concentrations would here be high enough that you can just do ELISA and see something with the naked eye?

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. 

2ChristianKl3y
This sounds to me like the kind of thing biology expertise to get right and potentially more equipment.