mono vs polyclonals; cross-reactions

Thu Mar 17 15:44:17 EST 1994

>High all!

>With indirect immunofluorescence double labelling experiments
>I have made good experiences using first ABs from different
>species and goat polyklonals as 2nd ABs (FITC and Cy3 conjugated)

>Now I have to set up a double labelling with two monoklonals
>from mouse differing only in the isotype: IgG1 contra IgG2b.
>There are lots of conjugated polyklonals that discriminate
>IgG1 and IgG2b, what about the cross reactions?

>The experiment should show the two antigens in one cell.
>There are some monoklonal 2nd ABs (only one red) available.
>I suppose  they are much more specific but expensive and all
>from rat. Could there occur other cross reactions e.g.
>Rat at IgG2b binding to a RatIgG2b at MouseIgG1??

>What system should I use monoclonal or polyclonal and are there
>other tricks to get good results in this case?

>Every comment is appreciated!

If your assay allows it, why don't you conjugate your Ab with
flourochrome (or whatever); you'll lose sensitivity and its a 
bit of a hassle, but you'll have no cross reactions. Otherwise
I think I'd go for the monoclonals, although I have had good
luck with some batches of polyclonals in similar situations, 
however, my system (DAS ELISA) could tolerate cross reaction -
I dind't mind a slight background. Try conjugation.

 Chris Rampitsch                       |       RAMPITSCH at BCRSSU.AGR.CA
 Dept. Plant Science, Univ. of BC      |       Phone (604) 494-7711
 Vancouver, British Columbia           |       Fax   (604) 494-0755
 Canada V6T 2A2                      _/\_
---------------------------------- __\  /__ ------------------------------------
"Most people don't act stupid;...  \__  __/ ..it's the real thing."  A.E.Neuman 

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