Posts Tagged ‘antibody’
Last Updated on Friday, 2 July 2010 10:09 Written by Administrator Friday, 2 July 2010 10:09
Question by teck kim: in ELISA, why the assay need secondary antibody labeled, why no just label the primary antibody?
Best answer:
Answer by Isopropyl_Dog
antibodies have two sides: a constant region and a variable region. So the primary antibody has a variable region that specifically binds to the antigen of interest… this primary antibody can be made naturally by the immune system of an organism. If a person is exposed to antigen X, the natural process in his body will create anti-X antibodies. So then we could take a sample of his blood and collect the anti-X antibodies (with affinity chromatography this is easy), purify them and use them in an ELISA to detect antigen X. Unfortunately the natural process does not label the antibodies… thats why we need secondary antibodies.
The secondary antibody specifically binds to the constant region of the primary antibody (all the primary antibodies will have similar constant regions) and has a detectable probe. So you can use the same secondary antibody with lots of different primary antibodies.
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Does anyone know which antibody pairs work with Enzyme-Linked ImmunoSorbent Assay (ELISA for short)?
Last Updated on Monday, 21 June 2010 05:33 Written by Administrator Monday, 21 June 2010 05:33
Question by Diddy: Does anyone know which antibody pairs work with Enzyme-Linked ImmunoSorbent Assay (ELISA for short)?
ELISA is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. It utilizes two antibodies, one of which is specific to the antigen and the other of which is coupled to an enzyme.
I need like a list of antibody pairs that can work with it.
We are working with rats that were injected with Saline & LPS.
Best answer:
Answer by Phillip R
You can check with: http://www.antigenix.com/Merchant2/merchant.mvc?Screen=CTGY&Category_Code=MP
There is a list of antibody pairs there that they sell.
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Tags: antibody, anyone, assay, elisa, EnzymeLinked, Immunosorbent, know, pairs, short, work | Posted under Elisa Assay Questions | 1 Comment
Last Updated on Sunday, 20 June 2010 01:01 Written by Administrator Sunday, 20 June 2010 01:01
Question by msako: Why would you use the same isotype of antibody in ELISA?
Situation: ELISA assay. Microtitre plate –> wells coated with trout serum. Have two antibodies (unknown A and B from mouse). Have to determine which one is the anti-trout. (the other one is anti-catfish). The antibodies are both of the same isotype. Secondary antibody is anti-mouse Ig.
Why do we use the same isotype of antibodies? Is it for negative control?
Best answer:
Answer by N E
It is a control in one sense, but there are several reasons you are using the same isotype. The primary antibody is almost always an IgG isotype, and this is true in your case also (because the secondary antibody is an anti-mouse IgG). The primary antibody is a mouse anti-trout antibody and is more specifically a mouse anti-trout antigen IgG antibody. The secondary antibody (used to detect whether or not there is any primary antibody bound to any antigens) is an antibody against mouse IgG (called anti-mouse IgG antibody). The reason you use the same isotype antibody in all cases as the primary is so you can reduce the complexity of the experiment and reduce the complications you have to worry about. For instance, if you used an IgG and an IgM in different dishes, and a secondary specific for each, then you would have to worry about different binding specificities to the antigen as well as for the binding of the secondary antibody and so you couldn’t directly compare the results. If you use the same IgG and same secondary in all cases, then you can generally directly compare your results, so the only changing factors are the amount and source of the antigen. It is also cheaper and more convenient this way.
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Tags: antibody, elisa, isotype, same, would | Posted under Elisa Assay Questions | No Comments