Question

The secondary antibody is attached to an enzyme (HRP) that chemically changes the enzyme-substrate, turning it from a colorless solution to a blue solution. If you ran an antibody detection ELISA with positive control wells, negative control wells, and experimental wells, predict which wells of your experiment should turn blue, which should remain colorless, and which wells you are not sure about and why.

Answer 1

In an antibody detection ELISA, the positive control wells should turn blue, the negative control wells should remain colorless, and the experimental wells may turn blue or remain colorless depending on the presence or absence of the specific antigen being tested.

Step-by-step explanation:

In an antibody detection ELISA, the positive control wells should turn blue because they contain the antigen that the primary antibody can bind to. The secondary antibody, attached to the enzyme, will catalyze the color change reaction. The negative control wells should remain colorless because they do not contain the antigen that the primary antibody recognizes. Without the antigen, the primary antibody will not bind, and subsequently, the secondary antibody-enzyme conjugate will not catalyze the color change reaction. The experimental wells may turn blue or remain colorless depending on the presence or absence of the specific antigen being tested. If the antigen is present, the primary antibody will bind, and the color change reaction will occur. If the antigen is absent, there will be no binding of the primary antibody, and the final mixture will remain colorless.

Answer 2

Positive control wells should turn blue, negative control wells should remain colorless, and experimental wells are not sure either they will turn blue or not.

Step-by-step explanation:

Basically the method of ELISA for antibody detection is known as indirect ELISA method.

In indirect ELISA the enzyme HRP is attached with secondary antibody and this antibody is attached with primary antibody along with antigen attached to the well bottom. When a substrate is added into the well the enzyme HRP present with secondary antibody act on substrate and turns the colorless solution into blue color.

Now, in positive control wells there must be present primary antibody and then secondary antibody along with HRP attached to this primary antibody then after substrate addition experiment color will change into blue color.

In negative control wells there is no primary antibody present due to this secondary antibody along with HRP unable to attach there in the experiment and wash out during washing step of ELISA and at the end there will be no color production because when we add substrate there will be no HRP enzyme to act on it.

In experimental wells, the sample is unknown and we don't know in this well or sample. the primary antibody is present or not that's why we are not sure about either this well solution change color or not. If primary antibody is present in the well then it's color will change into blue and if primary antibody absent in the sample then color will not change.