|Wells:||COSTAR flat bottom EIA 8-well strips, Catalog # 2580|
|Coating:||Add 100 µl of peptide or protein at a concentration of 5-10 µg/ml in 50 mM carbonate buffer pH 9.5. (Dissolve and adjust volume to 1 liter with distilled water.):Na2CO3 1.59 g
NaHCO3 2.93 g
NaN3 0.20 gIncubate at room temperature for 24 hours in a humid chamber.
|Blocking:||Remove coating solution, rinse twice with distilled water, and flip dry on paper towel. Add 200µl 0.1% BSA/PBS/0.02% thimerosal. Incubate for two hours at room temperature in a humid chamber.|
|Sample:||Remove blocking solution, rinse twice with distilled water, and flip dry on paper towel. Prepare serum dilutions using 0.1% BSA/PBS/0.02% thimerosal. Prepare the following dilutions: (Make 1:5 serial dilutions):Pre-immune serum control 1:1,000 / 1:5,000 / 1:25,000
Test bleed 1:1,000 / 1:5,000 / 1:25,000 / 1:125,000 / 1:625,000Add 100 µl of serum sample to wells. Run in duplicate.Incubate for two hours at room temperature in a humid chamber.
|Wash:||Remove serum dilutions and wash wells twice with distilled water. Flip dry.|
|Conjugate:||To each well add 100 µl goat anti rabbit-HRP conjugate. Dilute as recommended by manufacturer in 0.1% BSA/PBS/0.02% thimerosal. Incubate for two hours at room temperature in a humid chamber.|
|Wash:||Remove conjugate and wash wells three times with PBS/0.02% thimerosal/0.05% tween 20, and twice with distilled water. Flip dry.|
|Substrate:||Add 100 µl of TMB soluble substrate and develop at room temperature.|
|Stop:||Add 100 µl 1 N HCl to each well to stop color development.
Read immediately at A450.
Titer of the test serum is designated as the dilution which gives an OD reading of approximately 0.1 above background.
Next: Western Blot