ScFv ELISA data not right - (Nov/01/2007 )
I have run a few ELISAs using the ScFv's made from the RPAS system by Pharmacia(now GElifesciences) and my data is perplexing. There is no definitive curve, or trend that fits with the serial dilutions. I have tried using different titrations of the anti-E tag to no avail. I mostly get a rather flat line, or some random spikes. What could this be indicative of? 
My Protocol:
Protein our lab produced to coat the plate O/N at 4C. I block using BSA for an hour at RT before applying the ScFv in serial dilutions ranging from 1:10-1:10240, incubate for 1 hr at RT. I use anti-E tag at 1:1000 for an hour at RT, then apply anti-mouse IgG-HRP(Sigma) at 1:4000 for an hour at RT. I develop with ABTS at 10', 30' , and 60'. I wash 6X with PBST b/w steps.
My Protocol:
Protein our lab produced to coat the plate O/N at 4C. I block using BSA for an hour at RT before applying the ScFv in serial dilutions ranging from 1:10-1:10240, incubate for 1 hr at RT. I use anti-E tag at 1:1000 for an hour at RT, then apply anti-mouse IgG-HRP(Sigma) at 1:4000 for an hour at RT. I develop with ABTS at 10', 30' , and 60'. I wash 6X with PBST b/w steps.
I think your antigen/protein has lost it correct folding (denatured). This can also result false enrichment during panning/antibody selection. I've had similar problem and only realise after using the new batch of protein.
try increasing the Ab dilution from 1:10k till 1:200k.
and/or reduce the concentration of coating Ag to half or quarter.
observe the results......
gud luk