ELISA-Peptide Assay Protocol

A. Solutions and Reagents

1. Carbonate Buffer: 15 mM Na₂CO₃, 35 mM NaHCO₃, 0.2 g/L NaN₃ (pH 9.6). Use 1 μM synthetic peptide in carbonate buffer.
2. 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.
3. Wash Buffer: 1X PBS containing 0.05% Tween 20 (PBST)
4. Blocking Buffer: 10 mg/ml bovine serum albumin (BSA) in PBST
5. Antibody Dilution Buffer: 3% BSA in PBST
6. DELFIA Europium-labeled Anti-mouse IgG for mouse primary antibodies or Anti-rabbit IgG (PerkinElmer Life Sciences #AD0124) for rabbit primary antibodies.
7. DELFIA Enhancement Solution (PerkinElmer Life Sciences #1244-105)

B. Protocol

1. 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.
2. Wash plate three times 200 μl/well with wash buffer.
3. 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.)
4. Prepare appropriate dilution of primary antibody with antibody dilution buffer. Add 100 μl to wells and incubate at 37°C for 1 hour.
5. Wash three times with wash buffer.
6. 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.
7. Wash five times with wash buffer.
8. 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.