ELISA Protocols

The ELISA is a very flexible assay with a choice of formats, assay conditions, and detection methods.

We have included a few protocols below that can be used as a starting point for ELISA development in your laboratory, as well as basic ELISA buffer recipes. For more ELISA protocol guidance, please review the suggested reading.

Our specialized ELISA buffers resource page includes our complete collection of coating, blocking, washing, and dilution buffers, as well as ELISA substrates.

ELISA Protocols

• Direct ELISA with streptavidin-biotin detection
• Sandwich ELISA with direct detection
• Sandwich ELISA with streptavidin-biotin detection
• Quantitation of human cytokines by ELISA using AbD Serotec matched pair reagents

Buffers

N.B. Azide should not be added to any buffers that will be used directly with HRP-labeled antibodies because it will inactivate the enzyme.

PBS
8.0 g NaCl
0.2 g KCl
1.15 g Na₂ HPO₄
0.2 g KH₂ PO₄
Dissolve in 800 ml distilled water, adjust pH to 7.4, and then add more distilled water to a final volume of 1 liter. Sterilize by autoclaving and store at room temperature (RT).

TBS
8.0 g NaCl
0.2 g KCl
3.0 g Tris base
Dissolve in 800 ml distilled water, adjust pH to 8.0 with 1 M HCl, and then add more distilled water to a final volume of 1 liter. Sterilize by autoclaving and store at RT.

Coating
Carbonate-Bicarbonate
1.5 g Na₂ CO₃
2.93 g NaH CO₃
Distilled water, 1 liter, pH to 9.6.

Blocking
PBS or TBS Blocking Buffer w/ 1% BSA
Dissolve 1 g BSA Fraction V per 100 ml PBS (TBS) with gentle stirring and store at 4 C.

Sample Dilution and Complex Samples
Samples and standards are typically diluted in wash buffer.

Washing
PBST or TBST
PBS (TBS) with 0.05% v/v Tween® 20

Suggested Reading

1. Crowther, J.R. (2001) The ELISA Guidebook. Totowa (NJ) : Humana Press.
2. Harlowe, E and Land, D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbour (NY) : Cold Spring Harbour Press.