Basic questions.. - antibody titer (Nov/13/2006 )

Hi all,

I am making a polyclonal antibody. Now I got some blood sample, and was told to test the titer of this antibody... But, what is the titer of antibody and how to test it?



Thanks

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do a direct ELISA : coat your antigen, and test increasing dilutions of your serum.
There was already a topic about polyclonal antibodies and serum titration.

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That's quite good and may be helpful:
http://www.komabiotech.com/technical/protocol/ELISA.htm

(PS. There's way to calculate inflexion point with Excel if you have cuvre made by some good software like GraphPad Prism.)

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Thank you.

Now the problem is we don't have ELISA or purified antigen protein..... I understood that we are going to test the antibody by Western Blot, diluting serum and incubating with blot...

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Hi,
Before defining the method to titrate your antibody, you should know what applications you will use it for.
To go to the very beggining. To titrate your antibody (whether polyclonal or monoclonal) is to find out the maximum dilution (minimum amount of antibody) you need to obtain a value over the threshold of the negative control(s). Almost every immunological technique may be used for titration (i.e., ELISA, immunofluorescence, immunoprecipitation, WB, and so forth).ç
The antibody titer is usually expressed as the number of times you diluted the antibody to give the last positive signal in the assay. For example, if you diluted the antibody up to 1600 times, but you get a positive signal only after diluting it 200 times (dilution 1:200) the titer is 1:200 (also expressed 1/200 or 200, depending on the literature source).
The important issue is that you need to know the titer to:
1) Use the minimum amount of antibody that gives good signal to spare as much as you can (animal maintenance and antigen production is costly).
2) Use the minimum amount of antibody that gives good signal to avoid false positives (due to Ab aggregation) or false negatives (known as prozone effect) in excess of antibody. What really happens depends on the technique.
3) It is best to do a titration using the technical application that you will use the antibody for. As an example, although the principle is the same, titers usually differ when you titrate antibodies for fluorescence microscopy or fluorescence-activated cell sorting (FACS). More so if you test antibody titers with ELISA and then use it for immunoprecipitation.
You may want to read further. Then get "Antibodies: A laboratory manual" written by Ed Harlow and David Lane (Cold Spring Harbor Laboratory Press) or, "Current protocols in immunology", Edited by Wiley & Sons.
Hope this helps. Regards,
Gerardo

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Thank you very much Gerardo. I learnt a lot from your reply and will borrow the books later.

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