Protocol:
1) ADSORBTION OF ANTIGEN
a) Dilute Ag to 10 ug/ml in PBS.
b) Add 100 ul of Ag solution to each well.
c) Leave O.N covered with saran wrap, at 4 C.
2) BLOCKING
a) Wash unbound Ag by inverting the plates and flicking the wells dry.
b) Rinse by adding PBS to each well and inverting it again (use squirt bottle).
c) Repeat the rinse twice.
d) Add 100 ul of blocking solution to every well, leave 1 hr at room Temp or O.N at 4 C.
3) PRIMARY ANTIBODY
a) Add the antibody to be tested :
Sup of cells = 25 ul , mix well by pipeting up and down (10 times). serum, ascites = 1:100 and a serie of 1/5 dilutions. Do dilutions in blocking solution.
b) Leave 1 hr at room temp or O.N at 4¡C.
4) SECONDARY ANTIBODY
a) Wash unbound antibody 4 times with PBS + 0.1% Tween 20.
b) Add 100 ul of enzyme linked antibody to all wells. Do the appropriate dilutions in the Elisa buffer. (ex: HRP is 2000X).
c) Leave 1 hr at room temp or O.N at 4¡C.
5) SUBSTRATE
a) Disolve substrate in water.
b) Wash plate 4 times with PBS + 0.1% Tween 20 (use TBS instead of PBS for AP).
c) Add 100 ul of substrate to every well.
d) Watch color development. This could take from a few seconds to 20 min.
e) If needed, stop the reaction by adding 50 ul of 4M NaOH.
f) Read absorption in Elisa reader at correct wavelength (for HRP system 416 nm, for AP - 405 nm).
