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Help with the design of an ELISA experiment

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Help with the design of an ELISA experiment

Postby bemardus » Tue Apr 27, 2010 10:39 pm

Hi guys,

So I need help with an ELISA experiment. I need to determine the concentration of IgG in serum and what percentage of the cellular protein in said serum is a result of IgG. What I have to work with is mouse serum, mouse serum albumin, mouse IgG, rabbit anti-mouse IgG, goat anti-mouse IgG (biotin and FC-HRP), goat anti rabbit IgG (biotin and ap-conjugated), goat anti-mouse IgG HRP (gamma chain), rabbit anti mouse serum. I haven't an idea where this should begin. Plus what all these antibodies are used for. Any help would be awesome. Thanks.
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Postby Jillo725 » Tue Apr 27, 2010 11:27 pm

You'll be doing a sandwich elisa test. wikipedia it. it's got a great description and picture to go along with it. all proteins are accounted for in this test.
"There is no adequate defense, except stupidity, against the impact of a new idea."
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Postby bemardus » Wed Apr 28, 2010 4:18 am

Thanks Jillo725.

So guys am curious now though what all these antibodies are used for. After looking at wikipedia I have an idea of what to do but not what to use for each step. So I'm guessing I'd used mouse IgG as the capture antibody. Then rabbit anti-mouse IgG as the would be antigen. Following that I'd use goat anti-rabbit IgG to attach to the rabbit anti-mouse IgG. And I'd use goat anti-mouse IgG FC-HRP. Is this at all right?

With that what's the difference between ap-conjugate and biotin? And FC-HRP and gamma chain HRP? Which of these do I choose? And what's the purpose of mouse serum albumin and rabbit anti-mouse serum?

Sorry for all these questions. I'm totally lost here though. Thanks for the help though.
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Re: Help with the design of an ELISA experiment

Postby Jillo725 » Wed Apr 28, 2010 1:50 pm

Not sure about everything. just look for the definitions of those. it could be that the teacher listed extra proteins that are not used. I know that FC is the stem region of the antibody. I think (if i remember right) that the HRP is some kind of human protein....or fluorescence i can't remember, we're going back about a year and a half here =p
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Re: Help with the design of an ELISA experiment

Postby savvygirl » Fri May 07, 2010 2:13 am

hehe

Is this someone in Dr. Bob's Class?

:)
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