TBR test 2 biological sciences q 102

[2010premed]

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In the monoclonal antibody test, why is the urine sample completely rinsed from the well of the test kit before the enzyme-antibody complex is added?
A. It is done to prevent urine from binding to the first antibody, which it separates from the plastic wall.
B. Free hCG that has not been bound to the antibody may bind the enzyme-antibody complex.
C. Uric acid would denature the enzyme-antibody complex
D. The yellowish color of urine obscures seeing whether a color change has taken place, which happens when hCG binds the antibody in the test kit.

Answer: B,

why not D?

The passage gives this info : Any hCG in the urine is trapped by hCG antibody affixed to the plastic surface of the test kit well. A second antibody linked to a chromophore (an enzyme that changes color when bound) is then added to the sample to bind a different site on the hCG.

 

[Rabolisk]

Because you don't know that D is true. Don't assume.

 

[justhanging]

Because you don't know that D is true. Don't assume.

Am confused by this question too and this is not a good response. Check out TBR explanation:

The color change of a positive test does not occur until the second antibody has been added, so the yellowish color of urine would not interfere with a proper reading. Therefore, eliminate choice D. Any free hCG that has not bound the antibody is capable of binding the enzyme-antibody complex. This species (being unbound) may be lost in a subsequent wash, which would prevent a good reading. That makes choice B the best answer

First, would the yellow color of the urine interfere once the the enzyme is added? I thought that was the whole point to add the enzyme once hCG was bound so I don't get that explanation. I also don't get why B is right, the question asks why it necessary to was the well while the explanation seems to say the opposite, that any hCG would be washed away if not bound.

 

[BerkReviewTeach]

The passage says, "Monoclonal antibody tests use ELISA (enzyme-linked immunosorbent assay) to produce a color change indicating a positive result. Any hCG in the urine is trapped by hCG antibody affixed to the plastic surface of the test kit well. A second antibody linked to a chromophore (an enzyme that changes color when bound) is then added to the sample to bind a different site on the hCG. The monoclonal antibody test can detect lower levels of hCG, so it is faster than older tests. It is also less sensitive to external factors, including other hormones in the urine. Monoclonal antibody tests require five to thirty minutes to complete and are more than 95% accurate."

So let's first consider what should happen ideally and then what the impact of urine residue would cause.

The hCG in the urine sample binds the antibody bound to the plastic surface of the kit. This is not colored, so there is nothing yet to read. The residue should be rinsed away at this point, so that the second antibody can only bind hCG that has been bound to the antibody on the surface of the plastic. Once the second antibody linked to a chromophore (colored marker) binds, it changes color. This color change, and not the actual color, indicates the presence of hCG.

So if residual urine was there, and it's yellow color was present, it would simply distort (alter) the two colors (those of the bound and unbound second antibody), but a color change would still be observed (and that's what we are looking for). Let's say it normally goes from pink to blue if positive. With a little urine present, it might go from orangish-pink to greenish-blue, which could still be read as a color change.

If there is any free hCG that has not been rinsed away, it can bind the second antibody. This will reduce the amount that binds the hCG bound to the antibody on the plastic surface, which will reduce the amount of color change on the surface of the kit. The unbound hCG can be lost (rinse away, migrate away, or whatever), so even if it undegoes a color change, it won't be seen against the the plastic background. For the kit to work best, both the first antibody and the second antibody must bind hCG bound to the surface of the plastic, where we are looking for the color change.

 

[pm1]

The passage says, "Monoclonal antibody tests use ELISA (enzyme-linked immunosorbent assay) to produce a color change indicating a positive result. Any hCG in the urine is trapped by hCG antibody affixed to the plastic surface of the test kit well. A second antibody linked to a chromophore (an enzyme that changes color when bound) is then added to the sample to bind a different site on the hCG. The monoclonal antibody test can detect lower levels of hCG, so it is faster than older tests. It is also less sensitive to external factors, including other hormones in the urine. Monoclonal antibody tests require five to thirty minutes to complete and are more than 95% accurate."

So let's first consider what should happen ideally and then what the impact of urine residue would cause.

The hCG in the urine sample binds the antibody bound to the plastic surface of the kit. This is not colored, so there is nothing yet to read. The residue should be rinsed away at this point, so that the second antibody can only bind hCG that has been bound to the antibody on the surface of the plastic. Once the second antibody linked to a chromophore (colored marker) binds, it changes color. This color change, and not the actual color, indicates the presence of hCG.

So if residual urine was there, and it's yellow color was present, it would simply distort (alter) the two colors (those of the bound and unbound second antibody), but a color change would still be observed (and that's what we are looking for). Let's say it normally goes from pink to blue if positive. With a little urine present, it might go from orangish-pink to greenish-blue, which could still be read as a color change.

If there is any free hCG that has not been rinsed away, it can bind the second antibody. This will reduce the amount that binds the hCG bound to the antibody on the plastic surface, which will reduce the amount of color change on the surface of the kit. The unbound hCG can be lost (rinse away, migrate away, or whatever), so even if it undegoes a color change, it won't be seen against the the plastic background. For the kit to work best, both the first antibody and the second antibody must bind hCG bound to the surface of the plastic, where we are looking for the color change.

Hey BerkReviewTeach !
I am having some trouble with this question myself.
I chose A. I don't think it makes much sense since antibodies are very selective and shouldn't bind to urine.

However, I am still confused why B would be correct. Isn't the whole purpose of the ELISA test to get ALL the hCG present? I know that in the best case scenario it is to "sandwich" the antigen between the primary and secondary antibody. But, in the eventuality that the primary antibody didn't bind to hCG, isn't it better to have the secondary binding then not binding at all?

Thank you!