In a modified version of ELISA, a student first incubated antibody against the Pseudomonas aeruginosa exotoxin A (Pa-exotoxin A) with culture samples in a 0.5 mL tube to check for Pseudomonas contamination. Each antibody-culture mixture was then added to a microtiter plate whose wells were coated with Pa-exotoxin A. After removing and washing, an enzyme-conjugated secondary antibody and a substrate were added. The absorbance values at 450 nm for each of four samples A–D is:​

Select the option that arranges the samples from highest to least contamination:​

Correct option is A

In this modified ELISA, the principle is based on the competition between free Pa-exotoxin A (present in the sample) and the immobilized Pa-exotoxin A (on the microplate wells) for binding with the specific antibody.

If a sample contains more exotoxin, then more antibody will bind to the exotoxin in the tube and less antibody will be available to bind with the exotoxin coated on the well — leading to less color development and hence lower absorbance.

Thus, absorbance is inversely proportional to exotoxin concentration (contamination):

• Lower A₄₅₀ → More contamination

• Higher A₄₅₀ → Less contamination

Given this inverse relation, the correct descending order of contamination is:

• C (0.098) > D (0.220) > A (0.323) > B (0.582)

So, the correct order from highest to least contamination is:
C, D, A, B

Hence, the correct answer is (a).