A student performed an ELISA to detect anti-ovalbumin IgG in a serum sample. The experiment involved the following sequential steps: coating plates with ovalbumin, blocking with BSA, adding serum sample, adding anti-mouse-IgG-HRP, adding H₂O₂ + o-Phenylenediamine dihydrochloride (OPD), and adding H₂SO₄. The student made the following statements:

A. If the plates are not blocked with BSA, the specificity of the assay decreases.
B. If the plates are not washed between addition of serum sample and addition of anti-mouse IgG-HRP, the sensitivity of the assay decreases.
C. If the plates are not washed between addition of anti-mouse IgG-HRP and addition of H₂O₂ + OPD, the specificity of the assay decreases.
D. OPD is the substrate for the enzyme.
E. Without H₂SO₄, no colour is developed.

Which one of the following options represents the combination of all correct statements?

Correct option is C

The correct option is (c)

EXPLANATION-

OPTION  A - Blocking with BSA is critical because it covers uncoated surfaces on the plate to prevent nonspecific binding of antibodies or serum proteins. Without blocking, other proteins might bind nonspecifically, causing false positives and reducing assay specificity. This is correct.

OPTION B - Washing removes unbound serum antibodies. If washing is skipped, excess unbound antibodies remain, which can cause high background noise and reduce sensitivity (ability to detect true positives). This is correct. 

OPTION C - Washing after adding the secondary antibody (anti-mouse IgG-HRP) is important to remove unbound enzyme conjugate. Without washing, unbound enzyme can react with substrate, causing nonspecific color development, which reduces specificity. This is  correct.

OPTION D - OPD (o-Phenylenediamine dihydrochloride) is indeed a chromogenic substrate used for HRP . OPD is a substrate analog or chromogen, not the actual substrate that HRP uses to catalyze the reaction. The real substrate is hydrogen peroxide. This is incorrect​

OPTION E - H₂SO₄ is added to stop the enzymatic reaction and stabilize the color. Without H₂SO₄, the color would still develop, but it might continue to change or fade over time, making quantification unreliable. The color development occurs because of the OPD-HRP reaction, which happens before acid addition. This is incorrect  because Color is developed before adding H₂SO₄, which stops the reaction.

Option  (c)  as it covers the blocking and washing steps which are most critical to assay performance.
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