A research laboratory is developing a new ELISA for diagnosing acute leptospirosis. In a double-antibody sandwich ELISA (indirect ELISA format) to detect patient antibodies, the stages in order are:

• A Coat plate with patient serum → add enzyme-conjugated anti-leptospira → add substrate
• B Coat plate with anti-human IgG → add patient serum → add leptospiral antigen → add enzyme conjugate
• C Coat plate with leptospiral antigen → add patient serum → add enzyme-conjugated anti-human IgG → add substrate → read OD ✓
• D Add patient serum to plate → add leptospiral antigen → add substrate to detect antigen-antibody reaction

Explanation

In indirect ELISA for antibody detection: (1) the microtitre plate is coated with the specific antigen (leptospiral antigen/LPS/OMP), (2) patient serum is added and patient-specific antibodies bind the antigen, (3) unbound antibodies are washed away, (4) an enzyme-conjugated secondary antibody (anti-human IgG-HRP) is added, which binds any patient IgG captured on the plate, (5) after washing, a colorimetric substrate is added and colour development (proportional to antibody concentration) is read by spectrophotometer as OD450. This format detects patient immunoglobulin. The double-antibody sandwich ELISA is specifically for antigen detection, not antibody detection, involving two anti-analyte antibodies ('capture' and 'detection').

Reference: Ananthanarayan & Paniker's Textbook of Microbiology, 11th ed.

High-yield for: NEET PGINI-CETNExTFMGEUSMLEPLABMRCP

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