Question about ELISA assay

[circulus vitios]

I want to test for a particular antibody in a serum sample. I place the antibody in a well that has antibody specific antigen bound to the bottom. Then I add an enzyme labeled antibody that is specific to the serum antibody. This generates a colored solution, indicating the presence of the particular serum antibody.

My question is: what is the purpose of the antigen? If I'm adding an enzyme-labeled specific to the serum antibody I'm testing for, why do I need the antigen? I understand that my serum sample will contain many other antibodies that I don't care about, but shouldn't my ezyme labeled antibody be specific enough for this to not matter?

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[Leonardo Noto]

I was hoping that someone else would answer this--I "think" that I know the answer but I'm not a biochem Ph.D.

There are two main parts to a generic antibody. The Fc region is the "stalk" and it is more or less the same in all (generic) antibodies whereas the "fork" region is called the Fab region and it is highly variable among antibodies. This is b/c the Fab region is the portion that binds to the antigen.

It is difficult and expensive to make antibodies that are conjugated to an enzyme. One way to get more bang for your buck is to make enzyme-linked antibodies that are specific for the Fc region and, thus, that will bind to all antibodies (with some exceptions). For instance, if you want to test a patient's serum for reactivity to "A" blood antigen (as in type O blood, type A blood, type B blood, type AB blood) then you can coat your ELISA plate with A antigen and add some of the patient's serum to it. Then you wash the plate and add you enzyme-linked antibody that binds to Fc segments of all antibodies--if you get a reaction (color change) then you know that the patient has antibodies to"A" antigen and shouldn't receive type A or type AB blood. This is much easier than making an enzyme-linked antibody that is specific for the Fab segment of every antibody that you want to test for, especially since the Fab segment that binds "A" antigen may differ from patient-to-patient.

Hopefully someone else can give a definitive answer--like I said, I think that this is correct but I only answered b/c no one else had.

Dr. Leonardo Noto

 

[cheesier]

You have it a little mixed up. ELISA is generally used to test for an antigen, not an antibody. You need two antibodies because the first is for binding the antigen, and the secondary antibody is for producing a color change. You wash the sample between steps. I'd assume that the secondary antibody can be nonspecific and would any primary antibody that isn't washed away, indicating the presence of the angiten.

 

[Leonardo Noto]

You have it a little mixed up. ELISA is generally used to test for an antigen, not an antibody. You need two antibodies because the first is for binding the antigen, and the secondary antibody is for producing a color change. You wash the sample between steps. I'd assume that the secondary antibody can be nonspecific and would any primary antibody that isn't washed away, indicating the presence of the angiten.

My biochem is about a decade rusty and I'm not a pathologist, but I'm pretty sure that ELISA can be used to detect either an antigen or an antibody of interest, depending on what you're looking for and whether you're using the direct or indirect method. Here's a good tutorial that I found useful (very good, actually): http://www.sumanasinc.com/webcontent/animations/content/ELISA.html.

 

[cheesier]

I didn't realize that they used antigens to detect antibodies. I would've thought they would just use a different antibody, but it makes sense that they need to anchor it to the well so it doesn't wash away.

 

[Leonardo Noto]

Yeah, the ELISA stuff can drive you nuts thinking about it. With that said, it's definitely high yield material for the MCAT--the MCAT loves ELISA b/c it tests so many scientific concepts at the same time and requires second or third order thinking to answer the question. Hey, I've got a cool ELISA story for you. When I was in Iraq we had a guy with suspected malaria. We had limited lab capabilities but we did have ELISA-based malaria cards that have a sensitivity of about 99% or higher. We used the malaria cards twice and they were negative both times--but I know for a fact that the guy had malaria because I saw the intracellular ring forms on microscopic exam and b/c of his reaction to an antimalarial drug. What went wrong with the cards? Well, they had be transported through the desert in 120-130 degree heat without refrigeration--ELISA is protein-based and proteins denature in the heat!

Good luck on the MCAT, man!

 

[circulus vitios]

Thanks for the replies. 🙂

 

[SpecterGT260]

I want to test for a particular antibody in a serum sample. I place the antibody in a well that has antibody specific antigen bound to the bottom. Then I add an enzyme labeled antibody that is specific to the serum antibody. This generates a colored solution, indicating the presence of the particular serum antibody.

My question is: what is the purpose of the antigen? If I'm adding an enzyme-labeled specific to the serum antibody I'm testing for, why do I need the antigen? I understand that my serum sample will contain many other antibodies that I don't care about, but shouldn't my ezyme labeled antibody be specific enough for this to not matter?

Because you wash the solution out after each step. You need to make sure the Ab is stuck to something. Your labeled Ab (the one with the fluoroflore or enzyme) will be targeted against a specific Ig isotype or against a specific animal If depending on the kit. If you put in serum and don't wash, there will be all sorts of reaction happening because you have various isotypes against all sorts of Ag. You need to hold only the one with the appropriate Fab section in the well as the probe is specific for the Fc region of the serum antibody.

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[SpecterGT260]

My biochem is about a decade rusty and I'm not a pathologist, but I'm pretty sure that ELISA can be used to detect either an antigen or an antibody of interest, depending on what you're looking for and whether you're using the direct or indirect method. Here's a good tutorial that I found useful (very good, actually): http://www.sumanasinc.com/webcontent/animations/content/ELISA.html.

Yes. There are direct and indirect elisa tests.

Wanna find an Ab in a patient? Bind synthetic Ag to a plate, wash pt serum over it, and probe for any Ig that is bound.

Wanna find Ag? Bind specific Ab to plate, wash serum. Apply specific Ab again to serve as site for probe, and probe again.

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[SpecterGT260]

You have it a little mixed up. ELISA is generally used to test for an antigen, not an antibody. You need two antibodies because the first is for binding the antigen, and the secondary antibody is for producing a color change. You wash the sample between steps. I'd assume that the secondary antibody can be nonspecific and would any primary antibody that isn't washed away, indicating the presence of the angiten.

I disagree. Antibody titers are elisa type assays. About as common as the "turn your head and cough" exam.

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