ELISA Protocol:
| 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. |
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| 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. |
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| 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.
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