ELISA Peptide

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)

(DELFIA^® is a registered trademark of PerkinElmer, Inc.)

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 hrs 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 hr 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 hr.
5. Wash three times with wash buffer.
6. Add 67 ng/well DELFIA Europium-labeled Anti-mouse IgG or Anti-rabbit IgG, diluted in 100 μl/well antibody dilution buffer. Incubate at room temperature for 30 min, on gentle shaker.
7. Wash three times with wash buffer.
8. Add 100 μl enhancement solution and incubate at room temperature for 5 min. Read plate at 615 nm with an appropriate time-resolved plate reader.