Sandwich ELISA - IgY quantification - (Apr/07/2008 )
I'm trying to develop a protocol for a sandwich ELISA to quanitify IgY (IgG from chickens).
I have tried to do a checkerboard to determine my optimal concentrations, but everytime i get poor color development, very random across the plate.
I am coating with Sigma mouse monoclonal anti IgY in 0.05M carbonate buffer, ranging from 16ug/mL - 0.0078ug/mL, blocking with 3% BSA (i've tried milk and fish geltain too), plating my primary Ab (sigma chicken IgY) at concentrations 2000-15.6 ng/mL in PBS, and my seconday sigma rabbit anti-chicken conjugated to alkaline phosphatase.
I'm washing with PBS-tweeen (0.05%), coating overnight, blocking for 2 H, all other incubations 1H at 37deg C.
I can produce a standard curve using just the sigma IgY and secondary.
I've contacted Sigma, their technical support had no suggestions for me.
Anyone have any ideas where i am going wrong or directions i could check out?
We are using pure IgY as estandard, and anti-IgY of Sigma to make a direct ELISA for quantifying IgY in egg yolk, serum, and samples lyophilized from egg yolk.
The lineal range of standard curve is between 0-100 ng/ml. The only difference with your protocol is that we use the anti-IgY conjugated to peroxidase (Sigma A-9046). I'm not quite sure, but I seem to remember that the Tween (PBS-Tween) and alkaline phosphatase were not very friends!
I hope you serve
And are you able to produce a standard sigmoidal shaped curve using your method? does it work when using it to quantify samples in egg yolk, serum etc?
We compared an ELISA sandwitch and a direct ELISA, and with both we have a reproducible curve. Before do the ELISA's we thought that the direct ELISA would not be a good method, but the values of the reference controls with both methods are correct, 25mg/ml in egg yolk, and 6 mg / ml serum.