Sandwich ELISA

• Coating Solution: Antigen or antibody are diluted in coating solution to immobilize them to the microplate. Commonly used coating solutions are: 50 mM sodium carbonate, pH 9.6; 20 mM Tris-HCl, pH 8.5; or 10 mM PBS, pH 7.2. A protein concentration of 1-10 µg/ml is usually sufficient.
• Blocking Solution: Commonly used blocking agents are: BSA, nonfat dry milk, casein, gelatin, etc. Different assay systems may require different blocking agents.

Primary/Secondary Antibody Solution: Primary/secondary antibody should be diluted in 1X  blocking solution to help prevent non-specific binding. A concentration of 0.1-1.0 µg/ml is usually  sufficient.

• Antigen Solution: Sample antigen should be diluted in 1X blocking solution to help prevent non-specific binding. A concentration of 0.1-1.0 µg/ml is usually sufficient.
• Wash Solution: Typically 0.1 M Phosphate-buffered saline or Tris-buffered saline (pH 7.4) with a detergent such as Tween 20 (0.02%-0.05% v/v).

 Protocol

 Apply Capture Antibody

1. Add 100 µl capture antibody diluted in coating solution to appropriate wells.
2. Incubate 1 hour at room temperature.
3. Empty plate and tap out residual liquid.

 Block Plate

1. Add 300 µl blocking solution to each well.
2. Incubate 5 minutes, empty plate and tap out residual liquid.

 React Sample Antigen

1. Add 100 µl diluted antigen to each well.
2. Incubate at room temperature for 1 hour to overnight.
3. Empty plate, tap out residual liquid.

 Wash Procedure

1. Fill each well with wash solution.
2. Invert plate to empty, tap out residual liquid.
3. Repeat 3 to 5 times.

 Add Secondary Antibody Solution

1. Add 100 µl diluted secondary antibody to each well.
2. Incubate 1 hour at room temperature.
3. Empty plate, tap out residual liquid and wash as in step 4.
4. Give final 5 minute soak with wash solution; tap residual liquid from plate.

 React Substrate

1. Dispense 100 µl substrate into each well.
2. If desired, after sufficient color development add 100 µl of the appropriate stop solution to each    well.
3. Read plate with plate reader.

 Suggested filters for KPL Substrates:

• ABTS: 405-410 nm
• TMB: non-stopped 620-650 nm, stopped 450 nm
• OPD: non-stopped 450 nm, stopped 490 nm
• pNPP: 405-410 nm
• BluePhosÔ: 595-650 nm

 References:

• Perlmann, H. and Perlmann, P. (1994). Enzyme-Linked Immunosorbent Assay. In: Cell Biology: A Laboratory Handbook. San Diego, CA, Academic Press, Inc., 322-328.
• Crowther, J.R. (1995). Methods in Molecular Biology, Vol. 42-ELISA: Theory and Practice. Humana Press, Totowa, NJ.
• Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 553-612.