Why would you use the same isotype of antibody in ELISA?
Why would you use the same isotype of antibody in ELISA?
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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