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Antisera vs. Antibodies


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#1 skeuos

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Posted 05 November 2009 - 07:18 AM

Hi all,

For some reason while I can get antibodies (and even cell culture sups from hybridomas) to stain nicely, I can never get anything but background/non specific staining when using anti-sera. This happened most recently using a commercially available antisera that other people have published staining with. I have this trouble with OCT embedded sections as well as tissue culture cells on coverslips.

Does anybody do anything different (or know of reasons to do anything different) for antisera vs. affinity purified antibodies?

thanks!

#2 sgt4boston

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Posted 06 November 2009 - 04:17 AM

Antisera will have everything in it: specific (polyclonal) antibodies raised against antigen, antibodies that cross-react with similar antigens, antibodies that do not react with with the antigen at all.
Plus all normal proteins such as albumin, transferrin, complement, etc etc.

Affinity purified antisera will contain only the antibodies that react with the antigen; the antisera goes thru a purification processs where the antigen is on a column and the specific ab bind and then the column is washed and the antibodies bound to the antigen are eluted creating a specific affinity purified product (still polyclonal). There will be cross reacting antibodies in this mixture since it is polyclonal.

Your hybridoma will be monoclonal all the same antibody...depending upon how the original selection was made it can be very specific to the antigen or also have some cross reactivity as well.

#3 skeuos

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Posted 09 November 2009 - 07:57 AM

Thanks for the response. Yeah, I figure it's all that other junk that always gives me trouble, but I don't know of ways to improve blocking of those non-specific interactions. It's frustrating, because so many papers show antisera working beautifully. Are there any antisera-specific techniques or protocols that I'm just not aware of? Or does antisera just need to be used at ridiculously low concentrations/high dilutions to try to minimize background?

thanks!

#4 sgt4boston

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Posted 09 November 2009 - 10:58 AM

I do mostly immunoassays but the problems are similar. If you have sufficient antisera why not do an ammonium sulfate cut to remove most of the junk? It is so easy and simple to do.

You are (?) using a secondary ab-conjugate? Is it specific for the species IgG you have in your antisera; that is are the nonspecifics adsorbed out? If this conjugate cross reacts with non-IgG proteins of your antisera then you will have binding (it is specific since the ab recognizes proteins in addition to the IgG you are using for detection).

Thus, you have 2 things to consider: all the stuff in your antisera and the specificity of the 2nd ab-conjugate.




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