ELISA-Peptide Assay Protocol
A. Solutions and Reagents
- Carbonate Buffer: 15 mM Na2CO3, 35 mM NaHCO3, 0.2 g/L NaN3 (pH 9.6). Use 1 μM synthetic peptide in carbonate buffer.
- 10X Phosphate Buffered Saline (PBS): To prepare 1 L add 80 g sodium chloride (NaCl), 2 g potassium chloride (KCl), 14.4 g sodium phosphate, dibasic (Na2HPO4) and 2.4 g potassium phosphate, monobasic (KH2PO4) to 1 L dH2O. Adjust pH to 7.4.
- Wash Buffer: 1X PBS containing 0.05% Tween-20 (PBST)
- Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
- Antibody Dilution Buffer: 3% BSA in PBST
- DELFIA® Europium-labeled Anti-mouse IgG for mouse primary antibodies or Anti-rabbit IgG (PerkinElmer Life Sciences #AD0124) for rabbit primary antibodies.
- DELFIA® Enhancement Solution (PerkinElmer Life Sciences #1244-105)
(DELFIA® is a registered trademark of PerkinElmer, Inc.)
- Coat the wells of a 96-well microtiter plate with 100 μl of 1 μM synthetic peptide in carbonate buffer by incubating overnight at 4°C or for 2 to 6 hours at 37°C. If the peptide does not bind or absorb, try other buffers in the pH 4–8 range.
- Wash plate three times 200 μl/well with wash buffer.
- Block plate with 200 μl/well blocking buffer for 1 hour at 37°C. Wash plate three times with wash buffer. (May leave dry plate at 4°C for 1–2 months if desired.)
- Prepare appropriate dilution of primary antibody with antibody dilution buffer. Add 100 μl to wells and incubate at 37°C for 1 hour.
- Wash three times with wash buffer.
- Add 67 ng/well DELFIA Europium-labeled Anti-mouse IgG, diluted in 100 μl/well antibody dilution buffer. Incubate at 37°C for 30 minutes.
- Wash five times with wash buffer.
- Add 100 μl enhancement solution and incubate at 37°C for 15 minutes. Read plate at 615 nm with an appropriate time-resolved plate reader.
posted June 2005
revised September 2007