In article <5141dn$5er at sifon.cc.mcgill.ca>, Paula Lavery
<plaver at PO-Box.McGill.CA> wrote:
>Hi there!
>>I am currently having problems conjugating a hamster-anti-mouse CD3 mAb.
All the
>usual protocols are unsuccessful. Anybody out there have any hints or
magic tricks?
>Any and all suggestions welcome. Thanks!
>>Paula
I found some years ago that I could label anti-CD3 hamster Mabs (both I've
tried) by this procedure (that we use routinely for all Mabs):
Reductive Crosslinking of Antibody with Phycoerythrin
Reducing Buffer: 50 mM sodium phosphate, pH 6.0
1 mM EDTA
degass
Coupling Buffer: 50 mM sodium phosphate, pH 7.5
1 mM EDTA
degass
I. Maleimide labeling of PE
1) Exchange 3 mg PE into coupling buffer with PD-10 column
(pre-equilibrated with coupling buffer); chill on ice
2) Dissolve 1 mg sulfo-SMCC (Pierce) in 100 µl water; add 3 µl per mg PE.
3) Rotate gently for 1 hr at room temperature; foil covered
4) Isolate maleimide PE with PD-10 column (pre-equilibrated with coupling
buffer)
II. Reducing antibody with DTT
1) Antibody at 1 mg/1 ml in reducing buffer (can dialyze or use PD10 column
or use Centricon concentrator)
2) Add 1/50 vol 1 M stock soln of dithiothreitol (fresh in dist. water)
with mixing
3) Reduce for about 1 hour hour at room temperature; remove excess reagent
with PD-10 column equilibrated with coupling buffer
III. Forming crosslinked complex
1) Add 1 molar equivalent of SH-Ab to 1.5-2 molar equivalents of SMCC-PE
Example: for 0.5 mg antibody use 1.2-1.6 mg PE (more to taste)
2) Rotate for 3 hours to overnight
3) Add N-ethylmaleimide to block remaining free SH groups; 10 mg/ml DMF
stock soln (make fresh). Add 40 µg (4 µl) per mg Ab. Rotate 15-30 minutes
at room temp
4) Pass down PD-10 column (equilibrated with Tris/Saline) or dialyze to
remove excess N-ethylmaleimide
For anti-CD3, I've never been satisfied with biotin/fluorescein conjugates.
Hope it helps!
___________________________________________________________
Richard R. Hardy Member, Inst. for Cancer Research
Fox Chase Cancer Center Tel: (215) 728-2463
7701 Burholme Ave. FAX: (215) 728-2412
Philadelphia, PA 19111 E-MAIL: RR_HARDY at fccc.edu
