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You decide to design an assay for flu infection detection. HA protein is only present on the surface of influenza viral particles so you choose to design an ELISA assay to detect the presence of HA in human blood samples.

You will use THREE different antibodies for the assay. The experimental procedure is as follows:

Step 1. Incubate your assay plate with antibody (I) solution overnight to coat the surface of the assay plate with antibody (I). Remove antibody (I) solution and wash the plate extensively.

Step 2. Add blood samples to the coated plate and incubate for 30 min. Remove blood sample and wash the plate extensively.

Step 3. Add antibody (II) solution to the plate and incubate for 30 min. Remove antibody (II) solution and wash the plate extensively.

Step 4. Add antibody (III) solution to the plate and incubate for 30 min. Remove antibody (III) solution and wash the plate extensively.

Step 5. Add TMB substrate to the plate and monitor color development.

In your lab, you have the following reagents on hand:

Rabbit anti-HA polyclonal antibody

Mouse anti-HA monoclonal antibody

Rabbit anti-HA monoclonal antibody

Goat anti-rabbit polyclonal antibody conjugated to HRP

Goat anti-mouse polyclonal antibody conjugated to HRP

You decide to use Rabbit anti-HA polyclonal antibody to coat the surface of your assay plate.

What would be an appropriate negative control sample for your ELISA assay?

What is the expected colorimetric result from the negative control in your assay?

What would be an appropriate positive control sample for your ELISA assay?

What is the expected colorimetric result from the positive control?

Indicate whether or not the following antibody combinations could be used to successfully perform your assay.  If a pair can't be used successfully, indicate why not. (6pts)

Rabbit anti-HA polyclonal antibody as antibody (II) and Goat anti-rabbit polyclonal antibody conjugated to HRP as antibody (III)

Circle one:   yes     no

Mouse anti-HA monoclonal antibody as antibody (II) and Goat anti-rabbit polyclonal antibody conjugated to HRP as antibody (III)

Circle one:   yes     no

Mouse anti-HA monoclonal antibody as antibody (II) and Goat anti-mouse polyclonal antibody conjugated to HRP as antibody (III)

Circle one:   yes     no

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