Hi, I realized an ELISA to evaluate interactions of Lipopolysaccharide of a specific strain of Pseudomonas aeruginosa and SARS-COV-2 Spike Protein. I use like blank a well witout LPS to coat it, I continue with the other steps: addition of blocking solution, spike protein, anti IgG horseradish peroxidase, TMB substrate and stop solution. But in this well, color is produced.
I don't Know, what is my error.
Can you help me, please?
Sincerely
Lucy Villafañe
Your Method description is not very clear.
Just correct me if I am incorrect:
Blank well: No LPS coat; the block; then spike protein; then IgG anti-spike protein; then anti-IgG HRP. Wash between steps. Finally add chromogen.
Test well: LPS coat; the block; then spike protein; then IgG anti-spike protein; then anti-IgG HRP. Wash between steps. Finally add chromogen.
If you see no colour in blank but colour in test well - indicates spike binds to the LPS coat and is detected by the anti-spike protein IgG ELISA.
Test well: LPS coat, the Block, tiene spike protein-Fc, then IgG-HRP. Wash between steps. Finally add chromogen.
Blank: I don't cover the well with LPS, but I do the following steps. In the blank well the color is produced.
I think is the blocking buffer. I have used: PBS+BSA, PBS+skimmilk by 30 min, T:37°C
Do you get colour in both test and blank. Is there a difference in colour when you use BSA and milk block? Do you add detergent to your wash buffer (eg. Tween)?
Hi Normal wash buffer for ELISA consist of PBS containing a detergent. The detergent used are either PBS containing 0,05 % Tween 20 or PBS containing 0,05 % Triton X100.
Hi Is your research question: Does LPS bind to Spike protein? If so, do you have access to pure spike protein. Just thinking of easier methods to answer your research question.
Hi
Sorry for the delay in reply but got very tied up editing a thesis.
If you have access to pure spike protein then I suggest the following method. The reason for this suggestion is to keep assay as direct as possible to prevent potential artifacts.
Method:
1. Biotinylate the spike protein (kits are available from Thermo Fisher).
2. Coat plate with LPS. Control contain no LPS. Overnight at 4 C.
3. Block plate with 25 mg/ml casein in PBS (2 hours at room temperature).
4. Add biotinylated spike protein in PBS containing 0.05 % Tween 20. Incubate for 2 hours at room temperature.
5. Wash the plate 4 times with PBS containing 0.05 % Tween 20.
6. Add Avidin peroxidase conjugate (Sigma Aldrich/Thermo Fisher and many other reputable suppliers) as per manufacturer's instructions diluted in PBS containing 0.05 % Tween 20.
7. Wash the plate at least 4 times with PBS containing 0.05 % Tween 20.
8. Add substrate. Incubate. Stop and read plate.
For optimization with do checkerboard dilution ranges of LPS and the biotinylated spike protein.
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