Question

2. For the ELISA assay depicted below, what do the arrows indicate? Click here to enter answer 3. What would happen in terms of the colorimetric readout if you forgot to do the wash step after Step D (above)? Click here to enter answer-

Answer 1

Step A is known as Antibody coating in which some specific antibody is immobilized on high protein binding plates with an incubation period provided. protein used may be albumin.

Step B is known as protein capture in which dilutions are added to the wells which will be captured by bound antibodies.

Step C is called detection antibody in which a biotinylated detection antibody is added to enable the detection of captured protein.

Step D is called as formation of streptavidin enzyme conjugate in which streptavidin conjucated horsedash peroxidase is added to the wells and will bind to biotinylated antibodies.

Step E is the addition of a colorimetric substrate to the wells, which when catalysed by an enzyme will form a color solution.

Answer 3- Washing is neccesary so as to remove unbound antigen-antibody complexes. if in step D washing is not done it will lead to poor results as unbound molecules will not be washed. Hence causing a degradation in performance of the assay.