affinity determination of an antibody - (May/24/2008 )

Does anyone know a good procedure to determine the affinity from a specific antibody to his antigen?

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this can be done with a biacore system. at least i think this is what the people are doing at our institute with this machine

http://www.biacore.com/lifesciences/introduction/index.html

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To "his" antigen?
Biacore makes sense.
You can also look up this:
http://www.pharma.ethz.ch/institute_groups...ocols/bandshift

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Is -'his' antigen- a wrong word choice? Sorry for my english

What I wanted to know is, if there is an easy method to control the affinity and specificity of the antibody you buy. For example I'd like to use antigens against CD5, how can I control that the antibody is specifically for CD5 and not for another antigen? Could I control this with an ELISA?

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What are you going to use your antibody for? The best control you can use is an antibody that's been made in the same way (same species, same isotype) but against a different antigen. The specific one should give you a positive control, the other one wont.

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I agree that biacore is widely used for antibody affinity measurements, however, the results can be quite unreliable, especially for an unexperienced user. The main pitfall is the ease with which it ganerates data with little user-input (especially on modern systems), but it can be difficult to judge whether the obtained values reflect true affinity of the interaction.

I think that isothermal calorimetry is still the reference method for affinity constant determination.

Regarding specificity, you could try western blotting (providing that it works in WB), to see whether the antibody binds only to one protein of the expected size or do you see additional bands.

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