Protocol

Enzyme Linked ImmunoSorbant Assay (ELISA) 

  1. Dissolve immunizing peptide in 0.1 M sodium bicarbonate buffer, pH 8.4.  Adjust final concentration to 20 μg/ml.

  1. 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.

  1. Cover the wells of the plate with parafilm and allow peptide to bind to the plates overnight at 4 ˚ C.

  1. 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).

  1. Block the wells by adding 350 μl of a solution of 5% nonfat dry milk in TBST for 1 hour at ambient temperature.

  1. 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.

  1. Incubate for 2 hours at ambient temperature.

  1. Empty the serum from the wells and rinse four times with TBST.

  1. 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.

  1. Incubate for 1 hour at room temperature.

  1. Empty the serum from the wells and rinse four times with TBST.

  1. 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.

  1. To each well, add 100 μl of substrate solution.

  1. Incubate for 30 minutes at ambient temperature on an orbital shaker.

  1. Stop the reaction by adding to each well 100 μl 1N HCl.

  1. Incubate for 5 minutes at ambient temperature on an orbital shaker.

  2. Read OD at 450 nm.

MATERIALS

Costar EIA/RIA Plates (High Binding)     Cat No. 3369

Corning, Inc., Life Sciences

45 Nagog Park

Acton, MA  01720

800-492-1110

www.corning.com/lifesciences/

 

Blotting Grade Blocker (nonfat dry milk)     Cat No. 170-6404

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

 

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