Protocol |
Enzyme Linked ImmunoSorbant Assay (ELISA)
Dissolve immunizing peptide in
0.1 M sodium bicarbonate buffer, pH 8.4.
Adjust final concentration to 20 μg/ml.
Add 50 μl of the peptide solution to two rolls of wells (A-H) of a polystyrene microtiter plate for each antibody to be tested.
Cover the wells of the plate with parafilm and allow peptide to bind to the plates overnight at 4 ˚ C.
Empty the peptide solution from
the wells and rinse once with 10 mM Tris-HCl, pH 7.4, 150 mM sodium
chloride, 0.1% Tween-20 (TBST).
Block the wells by adding 350 μl of a solution of 5% nonfat dry milk in TBST for 1 hour at ambient temperature.
Empty the blocking solution and add 100 μl of 3-fold serial dilutions of the test antiserum starting at 1:100, 1: 300, 1: 900, through 1:218,700 diluted in blocking solution. To a second row, add 100 μl of control serum to each well using the same dilution scheme.
Incubate for 2 hours at ambient temperature.
Empty the serum from the wells and rinse four times with TBST.
To each well, add 100 μl of a 1:24,000 dilution of horseradish peroxidase conjugated secondary antibody that was made to the whole molecule of purified IgG from the test species.
Incubate for 1 hour at room temperature.
Empty the serum from the wells and rinse four times with TBST.
Prepare peroxidase substrate solution by adding 0.5 ml 0.2 M citrate buffer, pH, 3.3 and 0.25 ml 2 mg/ml TMB to 9.25 ml water. Prior to use, add 25 μl 3% hydrogen peroxide.
To each well, add 100 μl of substrate solution.
Incubate for 30 minutes at ambient temperature on an orbital shaker.
Stop the reaction by adding to each well 100 μl 1N HCl.
Incubate for 5 minutes at ambient temperature on an orbital shaker.
Read OD at 450 nm.
MATERIALS
Costar EIA/RIA Plates (High
Binding)
Corning, Inc., Life
Sciences
45 Nagog Park
Acton, MA
01720
800-492-1110
www.corning.com/lifesciences/
Blotting Grade Blocker
(nonfat dry milk)
Bio-Rad Laboratories,
Inc.
2000 Alfred Nobel Drive
Hercules, CA
94547
800-424-6723
www.bio-rad.com
Peroxidase AffiniPure
Rabbit Anti-Goat IgG (H+L) IgG
Cat No. 305-035-045
Peroxidase AffiniPure
Goat Anti-Rabbit IgG (H+L) IgG
Cat No. 111-035-045
Jackson
ImmunoResearch Laboratories, Inc.
872 West Baltimore Pike
West Grove, PA
10390
800-367-5296
www.jacksonlabs.com
KPL SureBlueTM TMB Microwell Peroxidase Substrate
Cat No. 52-00-01
KPL,
Inc.
910 Clopper Road
Gaithersburg,
MD 20878
800-638-3167
www.kpl.com