Help with the design of an ELISA experiment

[bemardus]

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.

[Jillo725]

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.

[bemardus]

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.

[Jillo725]

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

[savvygirl]

hehe

Is this someone in Dr. Bob's Class?