Enzyme labeled peptides

[SaintJude]

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Kaplan Passage-based question. Passage explains, EMIT, an assay to test for viral infection.

Full Passage on EMIT (no info missing): "EMIT is a competitive binding assay that relies upon an enzyme's inability to function when bound to an antibody. In this technique, a solution of antibody to a specific viral peptide is prepared. A sample of the host's blood, as well as an enzyme-labeled peptide, is added. A substrate that produces color when cleaved is added to the solution. "

Question: A laboratory uses EMIT to test a patient for the hepatitis B virus and the patient's test was negative. Which of the following would be present in this test?

A. Enzyme labeled antibodies for hepatitis B
B. Enzyme labeled hepatitis B viral peptide
C. Unlabeled hepatitis B viral peptide
D.Enzyme labeled antibodies for the hepatitis B antibody

😕 Can someone please explain the best strategy in tackling this question. I'm really lost

 

[Ssina]

So it seems to me they made antibody specific for viral peptide
Blood that they are mixing either has the viral peptide or doesn't have it
and they also mix the enzyme labeled peptide
and there is the enzyme that cleaves this peptide

so if the person is infected with the virus, then the viral peptide will compete for the antibody with the enzyme.... essentially we have less enzymes bound to the antibody.. and now they are free to cleave the peptide and produce color!

If the assay is negative, then that means there is no virus in the blood, so the enzyme binds to the antibody (no viral peptide to compete with the enzyme for the antibody) and now there is no enzyme to cleave the peptide to produce the color- so no color!

have to say the answers are weirdly worded, is the answer A!?

 

[MedPR]

Is the answer A?

Negative test = no HepB viral peptide = all antibodies bound to the enzyme-labeled peptide.

 

[SaintJude]

Oh, sorry! forgot the answer: It's B. But your response explaining the scenario makes a lot of sense. I didn't understand what was going on. It's not A, because there is no need to label the antibody with an enzyme. We don't want the antibodies cleaved by enzymes! The other key to this answer, is that even "negative tests" will have a little color produced, but just not as strong as a "positive" test. C'mon, how to know that? I know now, I guess...

Here is Kaplan's explanation:

In EMIT, a labeled and unlabeled peptide compete for antibodies. [Not until now do I understand that they labeled it with an enzyme like Ssina explained. Did not know you could label with an enzyme!] As the concentration of unlabeled peptide form an infected host increases, it is more likely to bind antibody than the [enzyme]-labeled peptide, leaving the labeled peptide free to catalyze the color-creating reaction. However, in an uninfected patient, the antibody will be mixed with only labeled peptide, since the host has not unlabeled [viral] peptide in the blood. Therefore, during an EMIT on a host uninfected with Hepatitis B, only antibody to Hepatitis B and labeled Hepatitis B viral peptide will be used, and little color produced.

 

[Ssina]

Oh, sorry! forgot the answer: It's B. But your response explaining the scenario makes a lot of sense. I didn't understand what was going on. It's not A, because there is no need to label the antibody with an enzyme. We don't want the antibodies cleaved by enzymes! The other key to this answer, is that even "negative tests" will have a little color produced, but just not as strong as a "positive" test. C'mon, how to know that? I know now, I guess...

loool well we are working with enzymes and substrates and competition... so it won't be clear cut!

Here is Kaplan's explanation:

In EMIT, a labeled and unlabeled peptide compete for antibodies. [Not until now do I understand that they labeled it with an enzyme like Ssina explained. Did not know you could label with an enzyme!] As the concentration of unlabeled peptide form an infected host increases, it is more likely to bind antibody than the [enzyme]-labeled peptide, leaving the labeled peptide free to catalyze the color-creating reaction. However, in an uninfected patient, the antibody will be mixed with only labeled peptide, since the host has not unlabeled [viral] peptide in the blood. Therefore, during an EMIT on a host uninfected with Hepatitis B, only antibody to Hepatitis B and labeled Hepatitis B viral peptide will be used, and little color produced.

well all that was for nothing, eh! yeah as I mentioned their wording was weird to me

 

[pdeco1]

Can someone explain how we would know that the enzyme-labeled peptide was a viral Hep. B peptide?

If the host is infected. How does the enzyme compete with the viral peptide for the antibody if the enzyme is already bound?

Does "labeled" just mean that the enzyme is attached to the peptide or that it has the correct conformation to attach?

Thanks.

 

[MedPR]

Wow I completely forgot I answered this (tried to) thread before. Just rereading it now, answer B is the obvious answer (I guess that shows how far I've come in the last 2 months 😉)

The passage says:

A sample of the host's blood, as well as an enzyme-labeled peptide

So, there's no Hep B virus, and since there's no Hep B virus, there is also no Hep B antibody. C is wrong because "unlabeled hep b peptide" would be there only if the patient had hep b.

 

[SaintJude]

They tell you that EMIT is a competitive binding assay. This means that in EMIT, you basically have 2 nearly identical entities vying for one antibody. Like 2 twins vying for the princess. Princess = antibody.

The first contender is the viral Hep B peptide. This contender however may be no-show (depending upon whether the host is infected or not) And if it's a no-show, the 2nd contender sweeps in & binds to the antibody.

The 2nd contender is a constant in EMIT: it's the enzyme-labeled peptide. (Since this is a test for Hep B, you can assume that the scientist would be clever enough to include the relevant peptide--the Hep B peptide) When I first read this question, I had no clue why one would want to label a peptide with an enzyme. But it's actually a genius move. The passage tells you that the enzyme will be unable to function if it is bound to an antibody. But as a twist, they leave it up to you to understand that the enzyme's function is to produce the color by cleaving the added substrate. (In retrospect, who else would do it?)

So, back to scenario. The antibody is left at the altar, b/c the viral peptide B is not available. What's the antibody to do? It will bind to the enzyme-labeled peptide b/c there's no one else to compete for the spot. And once the enzyme is bound to the antibody it can no longer cleave the substrate and thus produce color. The test comes back as "negative". Had the viral peptide B of the host been available, both contenders would have vied for a spot with the antibody. So this would mean, that the enzyme-labeled peptide would retain more of it's ability to go about its original function and thus produce a stronger color.

I hope that answer wasn't overkill, but I partly wrote it for myself as well--b/c I may have to look at it again in a month.

 

[MedPR]

Your explanation is great. The one thing I'm not seeing is what substrate the added enzyme would be cleaving?

 

[SaintJude]

Your explanation is great. The one thing I'm not seeing is what substrate the added enzyme would be cleaving?

They don't tell you what compound the substrate is. But they say "A substrate that produces color when cleaved is added to the solution."

They're really saying the "Enzyme cleaves a substrate and this cleaved substrate then releases color"

Is that what you're asking?

 

[MedPR]

They don't tell you what compound the substrate is. But they say "A substrate that produces color when cleaved is added to the solution."

They're really saying the "Enzyme cleaves a substrate and this cleaved substrate then releases color"

Is that what you're asking?

Yea I think so. So color production is the indicator of a positive test; no color is a negative test. The test involves adding BOTH an antibody and a competitive inhibitor (for that antibody) of the virus being tested for, HBV in this case. If there is no HBV in the patient, the antibody you added has no choice but to bind with the enzyme (the inhibitor) that you added. Antibody bound to the enzyme = inactive enzyme = enzyme won't cleave the substrate (whose identity doesn't matter in this case) = no color = negative.

Got it, thanks!