I am using ELONA technique (analog of ELISA) to detect recombinant SARS-CoV-2 RBD spike protein in nasal fluid. I got confused as to what background or blank to use for my experiment. Which of the following, would you recommend as appropriate to use as the background/blank (for subtraction from other wells)
1. DI water
2. Dilution buffer (Na2CO3)
3. Nasal fluid diluted in Na2CO3 without the spike protein.
Thank you, I await your suggestions.
I would use nasal fluid from someone who was known not to be infected now or in the past with the virus, if it is available. Even better might be to use a pooled specimen from several such individuals to get a more representative sampling of the background.
Since I would suppose that this fluid would vary in concentrations of various components, such as total protein, between samples, the results should be normalized to (i.e., divided by) the protein concentration of the sample.
I will select number 2, using a pooled sample from many healthy individuals who are not infected previously or at the present time. Meantime to prepare the pooled sample of the nasal fluid I will mix a volume of each sample that contains a fixed amount of the protein (say 50 micrograms).
Good luck.
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