Design an experiment using Western blot to distinguish between total human IgG, IgM, and IgA responses to an antigen on the membrane while minimizing cross-reactivity between detection antibodies. Which combination of reagents and steps is the most appropriate?

Incubate one membrane with patient serum and a mixture of all isotype-specific secondary antibodies simultaneously so that total antibody response is detected in a single lane and relative isotype contributions can be deconvolved computationally afterwards without separate membranes.

Probe identical duplicate membranes with patient serum and then detect separately with isotype-specific labeled secondary antibodies (e.g., anti-human IgG-HRP on membrane A, anti-human IgM-HRP on membrane B, anti-human IgA-HRP on membrane C), including secondary-only controls for each membrane to monitor non-specific binding; this allows isotype-specific mapping without cross-reactivity between secondaries.

Run the serum sequentially on the same membrane with increasing concentrations of a universal anti-human secondary antibody to progressively reveal isotype-specific signals; this approach assumes later incubations will emphasize less abundant isotypes selectively.

Use fluorescently labeled primary antibodies against IgG, IgM, and IgA directly added to the serum before incubation, avoiding secondaries entirely because direct labeling of every isotype in complex serum always yields higher specificity and eliminates cross-reactivity concerns.