Design an FA-based workflow to determine if a patient has a current bacterial infection versus only past exposure. Which of the following experimental designs would best distinguish the two conditions?

Only perform a Western blot to check for protein bands because FA cannot differentiate current versus prior exposure; use the most sensitive antibody and omit washing to preserve weak interactions.

Run a direct FA on patient sample smear using fluorescent primary antibodies to detect current antigen on a slide, and in parallel run an indirect FA using laboratory antigen incubated with patient serum and fluorescent secondary antibody to assess past exposure, including appropriate positive and negative controls and stringent wash steps between antibody incubations.

Use a hemagglutination assay alongside a direct FA but exclude positive controls and skip secondary antibodies entirely, as the presence of any fluorescence or agglutination is sufficient to conclude both active infection and immunity.

Perform two indirect FAs: one with heat-inactivated patient serum at a single dilution and another using patient urine as a substitute for serum, both detected with fluorescent tertiary antibodies but without direct patient slides, relying solely on a single negative control.