Protein Protocols Protein Protocols

Enhanced Chemiluminescence 1093
is made by diluting AFP standard serum (Dako cat. no. X900 or Boehring Diagnostics
[Westwood, MA] cat. no. OTOD 02/03) in human serum containing 0.05% (w/v) sodium
azide. Store frozen as 1-mL aliquots.
13. Peroxidase-conjugated anti-AFP (Dako cat. no. P128) diluted 1/1000 in assay diluent.
Make up fresh for each assay batch.
14. Working enhanced chemiluminescence substrate solution (see Note 9)—either:
a. p-iodophenol-enhanced substrate: 1.25 mM luminol, 4 µM p-iodophenol, 2.7 mM H 2O 2).
Add 15 µL of stock hydrogen peroxide and 40 mL of p-iodophenol stock solution
to 50 mL of stock substrate: solution,, and mix well. Make up daily using high-quality
deionized water (see Note 3), and store in the dark when not in use, or
b. p-Hydroxycinnamic acid-enhanced substrate (1.25 mM luminol, 30 µM p-hydroxycinnamic
acid, 2.7 mM H 2O 2). Add 15 mL of stock hydrogen peroxide and 1 mg
of p-hydroxycinnamic acid (alternative names: p-coumaric acid or 4-hydroxycinnamic
acid, e.g., Aldrich [Gillingham, UK] cat. no. 2,320-7) to 50 mL of stock substrate
solution. Mix for 30 min before using. Make up daily using high-quality deionized
water (see Note 3), and store in the dark when not in use.
3. Method
1. Coat the wells of a microtiter plate with 100 mL of anti-AFP (1/1000 in coating buffer).
Allow the protein to bind for either 2 h at room temperature or overnight at 4°C (see Note 10).
2. Empty the wells, and wash off any unbound antibody by filling each well with PBS and
shaking the plate to re-empty. Repeat the wash and block unbound sites by incubating with
200 mL/well of blocking solution for 30 min at room temperature.
3. Empty the wells, and wash the plate twice (as described in step 2) with PBS-Tween to
remove any unbound albumin.
4. Prepare 1/20 dilutions of samples, standards, and quality control specimens using assay
diluent, and add 150 µL to each of the microtiter plate wells. Cover the plate with plastic
film, and incubate at 37°C for 1 h.
5. Empty the wells, and wash the plate three times with PBS-Tween. Shake the plate over a
sink to ensure complete removal of wash solution. Add 150 µL of working conjugate to
each well, cover, and incubate at 37°C for 1 h.
6. Empty the wells, and wash the plate three times with PBS-Tween made using high-quality
deionized water (see Note 3).
7. Add 150 µL of enhanced chemiluminescent substrate to each well in the same order and
preferably with the same timing as used by the plate reader. Allow at least 2 min for the
light output to stabilize before reading the plate.
8. Obtain unknown specimen results either by reading off a plotted calibration curve or use a
computer program to calculate from a fitted curve.
4. Notes
1. Peroxidase is inactivated by anions and certain antimicrobial agents, including azide, cyanide,
and thiomersal. Antimicrobial agents may be present in concentrated enzyme label
solutions and assay buffers at typically active concentrations, but must not be present in
wash solutions or substrate. The latter reagents must be freshly made each day from concentrated
stocks.
2. Powerful oxidizing or reducing agents may interfere with any peroxidase detection reaction
by inactivating peroxidase, oxidizing the substrates or reducing the oxidants in the
reagents. There are potentially many of these in the laboratory environment, including
chlorine in water, disinfectants, paper dust, laboratory coats, skin, and so forth. Care should
be taken to avoid contamination of individual assay wells or equipment.