ELISA Buffers & Recipes
A blocking buffer is a solution of non-specific protein, mixture of protein or compound that non-specifically binds to surfaces of the plate that are not occupied by the coating protein.
Blocking buffers can be effective if they improve the sensitivity of an ELISA assay through reducing background and signal to noise ratio.
Ideal blocking buffers will be to all non-specific sites, thus eliminating background, reducing non-specific signals without obscurring the analyte epitope for antibody binding.
Standard ELISA blocking buffer:
- Phosphate buffered saline (PBS) with 1% w/v BSA
An ELISA coating buffer is used to immobilize proteins/analytes or antibodies on microtitre plates.
Key factors in immobilization of analytes/antibodies on to microtitre plates can be the pH of the coating buffer. Selecting a coating buffer between pH 7.4 and pH 9.6 can have an affect on the steric structure of protein/antibody/analyte binding and thus affect their immobilization.
Testing of coating buffers can help increase mobility and performance of immobilized antibodies.
Standard coating buffer recipe:
- Na₂CO₂ 1.5g
- NaHCO₃ 2.93g
- Distilled water, 1 liter, pH 9.6
Recommended substrates and stop solutions
In the presence of HRP (Horseradish peroxidase) conjugated enzymes, TMB and peroxide react to produce a blue bypproduct which has a maximum absorbance at 605 nm. The colour intensity produced by HRP activity is proportional to the levels of analyte in the ELISA assay.
Following TMB (3,3’,5,5’ – tetramethylbenzidine) incubation a stop solution of 0.16M sulfuric acid is added to halt the reaction.
The amount of stop solution added should be equal to the amount of TMB substrate added to each well of the ELISA plate (typically 50 – 100 uL per well).
Following addition of sulfuric acid stop solution. The color changes from blue to yellow, which stabilizes the color development and allows the accurate measurement of intensity at 450nm using a spectrophotometer.
PNPP (p-nitrophenyl phosphate)
pNPP is a chromogenic substrate for alkaline phosphatases.
pNPP for use with alkaline phosphate-conjugated antibodies. Alkaline phosphatase catalyzes the hydrolysis of pNPP to pNP. This results in the production of a yellow phenolate which has a maximal absorption at 405nm. pNNP is sensitive to light and thus should be protected.
The goal of an ELISA wash buffer is to remove any signaling altering debris and preserve ELISA components. ELISA plates are washed prior to the addition of standards and samples, following the addition of detection antibody and following the addition of HRP conjugate antibody.
Both Tris based and PBS based wash buffers can be used in ELISA protocols.
PBS Wash Buffer
- PBS containg 0.05% V/V TWEEN-20
Tris based Wash Buffer
- Tris Buffered Saline (TBS)
- 6.06 g Tris Base
- 8.2 g NaCl
- 6.0 ml 6 M HCl
- 1 L of distilled water
- pH should be 7.2 to 7.8, conductivity should be 14,000 to 16,000
- 1 L of TBS
- 5 ml of 10% Tween 20