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You need to use protein-protein conjugation techniques. These
should be mild and work such that you avoid large aggregates.
We have used protein crosslinking reagents for making these
conjugates. The crosslinking reagents SATA, SPDP, and SMPB are all
available from Pierce (USA). We use SATA to substitute lysine residues
on R-PE. Once substituted the R-PE can be stored for long periods of
time and when required can be treated with hydroxylamine to expose a
reactive sulfhydryl bond. We treat the pure mAb with either SPDP or
SMPB each of which substitutes to lysine residues on the mAb. The
SPDP or SMPB on the mAb can then react with the free sulfhydryl residue on
the SATA substituted PE. In the case of SPDP the complexes form by
di-sulfide linkages. In the case of SMPB the complexes form a much
stronger thioether bond. In our hands there is usually a 10-20% recovery of
useful mAb-PE conjugates from the reactions, so you have to deal with
some reasonable losses in production. You will require size exclusion
chromatography or HPLC/FPLC to isolate the conjugates. We have used this
method to substitute both mouse and rat mAb.
I presume that you have a unique mAb that you want to substitute, because
if the mAb-PE is commercially available you're better off to buy it.
Working out the method will consume some time and the major expense of
R-PE which you will have to buy in 5-10 mg quantities.
I recently published a methods chapter describing these mAb-protein
conjugation techniques. If your interested I'll send you a copy. The
technical people at Pierce may also have some good suggestions.
Please send me any useful methods that you receive from others, if they
are not posted to the cytometry list.
Good luck,
Denis Snider
Dept. of Pathology
McMaster University
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On Tue, 23 Jan 1996, Kathy McKinnon wrote:
> Does anyone have a good procedure for coupling R-PE to antibodies. I have a
> procedure for FITC, but it seems that the chemistry for R-PE is different. I
> would appreciate any procedures and helpful hints.
>
>
> Thanks,
>
> Kathy McKinnon
> Cell Biology
> Human Genome Sciences, Inc.
> kathy_mckinnon@hgsi.com
>
>
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