Phage ELISA reproducability

Stephanie Gunn steph at ichr.uwa.edu.au
Tue Jan 16 21:27:58 EST 2001


I'm having troubles replicating "phage ELISAs".  Briefly, I'm using a
protein 3 display phage display system, and after biopanning would like to
use ELISA to screen my clones.

I am currently using Nunc Maxisorp plates, coating overnight with 1ug/ml
target antibody in a carbonate coating buffer.  The plates are blocked with
1% casein in PBS (I have also tried blocking with BSA and skim milk powder;
casein seems to give the best blocking), then washed three times with PBS.
I then add phage supernatant (Prepared by growing 2ml of phage in a 1:100
dilution of overnight host cells in 2xYT.  The culture is grown for 5 hours
at 27C, 225rpm and the bacteria cleared by two spins).  This is reacted for
2-3 hours at RT on an ELISA plate shaker.  The plates are then washed 6-8
times with PBS.  I then add a 1:5000 dilution of anti-M13 mAb (Pharmacia)
in 1% casein, which is allowed to react 1 hour at RT, again on the ELISA
plate shaker.  Plates are washed 6-8 times with PBS + 0.05% Tween.  K-Blue
TMB substrate is added, the plates developed, the reaction stopped by
addition of 1M H2SO4.

The major problem I'm having is that the results do not seem to be
replicable.  For example. I can actually add the same sample to multiple
wells and get readings that vary as much as 0.5OD between the wells.

What am I doing wrong?

Thanks in advance,

Stephanie Gunn.

___________________________________________________________
Stephanie Gunn
PhD Student
Molecular Biology Divison
Institute for Child Health Research
(Company Limited by Guarantee ACN 009 278 755)
Subiaco, Western Australia, 6008
http://www.ichr.uwa.edu.au

Postal address: PO Box 855, West Perth W.A. 6872 Australia
Ph: 61 8 9489 7874  Fax: 61 8 9489 7700
email: 	steph at ichr.uwa.edu.au
___________________________________________________________



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