Peptide ELISA

wayne conlan wayne.conlan at nrc.ca
Tue Feb 1 14:01:46 EST 2000



Tom Barr wrote:

> Hello all,
>     I've been struggling for some time now trying to measure mouse
> antibody responses to  synthetic peptides which have been  coupled to
> various carrier proteins. Does anyone out there have experience in
> peptide ELISAs who could perhaps give me some helpful advice? I've tried
> conjugating with glutaraldehyde and EDC to OVA for coating, UV treatment
> of plates, high pH buffers, poly lysine and even drying the antigen onto
> the plate... all with no success. Any advice/tips would be greatly
> appreciated...
> Thanks in advance,
>
> Hi Tom:

                In the past, I've raised rabbit antisera to peptides
conjugated to KLH.  To coat the uncojugated peptide I used "high activated"
PVC plates from Flow laboratories.  I'd dissolve the peptides in 50% acetic
acid then immediately dilute them to the required concentration in carbonate
buffer and dispense into the wells.  Then, we'd let the peptide dry onto the
plates by incubating them overnight at 37C.  Just about all antisera reacted
with their respective peptide by this method though with widely varying
titres.  For more details check out Conlan et al, 1989. J. Gen. Microbiol,
vol 135, 3219-3228.
Good luck.

Wayne.






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