Indirect ELISA troubleshooting: If the patient serum step is accidentally omitted while all other steps are performed as usual, what outcome should you expect regarding secondary anti-human Ig conjugate binding and optical density (OD) readings?

Introduction / Context:
An indirect ELISA (enzyme-linked immunosorbent assay) typically detects patient antibodies by immobilizing antigen, adding serum (primary antibody source), then detecting with enzyme-labeled anti-human immunoglobulin. Understanding what happens if the serum step is skipped is essential for troubleshooting low signal problems and preventing false negatives.

Given Data / Assumptions:

• Solid phase is coated with specific antigen.
• Patient serum (source of primary human antibodies) is omitted.
• An enzyme-conjugated anti-human Ig (secondary antibody) and substrate steps still occur.
• Proper blocking and washing are performed.

Concept / Approach:
In indirect ELISA, the secondary antibody requires bound primary human Ig on the plate to provide specific anchoring via Fc binding. Without primary antibody, the conjugate lacks a specific target and should be removed during washes, yielding baseline optical density comparable to the negative control.

Step-by-Step Solution:
Omitting serum removes the primary antibody layer.Secondary anti-human Ig has no specific Fc region to bind on the plate.Washing eliminates unbound conjugate, preventing color development.Substrate addition therefore produces OD near the blank/negative control.

Verification / Alternative check:
Run a control well lacking serum in parallel; OD should approximate the background from blocked antigen-only wells, confirming the interpretation.

Why Other Options Are Wrong:

• Anti-human Ig-conjugate binding non-specifically to polystyrene is minimized by blocking; significant OD from nonspecific binding is unlikely.
• Selecting only one of the two correct statements is incomplete; both outcomes occur together.

Common Pitfalls:
Assuming the conjugate binds directly to antigen; in indirect ELISA it recognizes human Ig, not antigen.

Final Answer:
Both (a) and (b)