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We tried conjugating two rat monoclonal antibodies (IgG2a-kappa,
IgG2a-lambda) with TRITC (Sigma T-5656, lot 20H5010). Both lost
over 90% of their binding activity by FACS analysis of the direct
staining (514 nm excitation on a FACStar -- there was some signal
at high concentrations). The conjugations were run at 0.6-1.0 mg
Ig/ml, 12 micrograms TRITC (from DMSO stock) per mg Ig (13
ug/ml), 30 min at room temperature. A550/A280 ratios were 0.24
and 0.22. We're in the process of titrating these conjugates
indirectly to confirm the loss of binding activity.
Similar conjugation of one of these antibodies (IgG2a-kappa) with
FITC produced a specific activity loss of only approximately 16%
The reaction was at 0.6 mg Ig/ml, 125 micrograms FITC (from DMSO
stock) per mg Ig (71 ug/ml), 25 min at RT. F/P was 4.1.
Earlier, we conjugated a mouse IgG1 with TRITC with good results.
This was Molecular Probes mixed isomers lot 10B-1, 8 micrograms
TRITC (from DMSO stock) per mg Ig, 10 ug/ml, 30 min at room
temperature. The IgG1 was at 1.2 mg/ml during conjugation.
Here, there was no loss of specific activity (in fact it appeared
to increase). A550/A280 was 0.23.
I've considered:
1. TRITC conjugates to an amino near the binding site,
inactivating the antibody. Seems unlikely since FITC didn't do
this to one of the antibodies (second now being tested), and two
different antibodies suffered the same fate.
2. I've horribly over-conjugated with TRITC but not with FITC.
Seems unlikely since my A550/A280 ratios are OK.
3. Sigma TRITC is death to antibodies, but Molecular Probes TRITC
they like.
(This note is being emailed to the cytometry mailing list,
and FAXed to Molecular Probes.)
--
/* - - - - - - - - - - - - - - - - - From - - - - - - - - - - - - - - - - - -
Eric Martz emartz@titan.ucc.umass.edu Professor of Immunology
Voice 413-545-2325, FAX 413-545-1578
Morrill IVN Rm 203, Dept Microbiol, Univ Mass, Amherst MA 01003 USA
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - */
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