I've never done your procedure so I'm speaking somewhat
naively here. However, I've done a fair amount of
antigen-antibody ELISAs in the past and have noted that I
would get variable ODs if I allowed the wells to dry too
much after they were washed. My problem cleared up by
simply working more quickly in getting the next reagent into
the wells after the wash steps were completed. If you're
using a plate washer you should also check to be sure that
it is washing consistently from well to well, and from wash
cycle to wash cycle.
I hope this is a simple help for your problem.
Stephanie Gunn wrote:
> 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> ___________________________________________________________