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ELISA Optimization Tips:
BLOCKING OPTIMIZATION
Learn how to optimize your ELISA blocking technique by reading this guide. It describes selecting right blocking buffers and a variety of available buffers including detergent and protein blockers. Find the ELISA handbook below to get more tips on ELISA optimization.
What is Blocking Buffer in ELISA?
The process of coating an ELISA plate relies on the passive binding activity of the solid phase, which immobilizes biomolecules on the well surface. Without appropriate blocking, the plate would bind the detection antibody alongside the antigen or detection antibody, resulting in high background signal and low sensitivity. Exposing the plate to a blocking buffer after coating causes the free binding sites on the well bottoms become saturated, removing the possibility of nonspecific binding and greatly improving the signal-to-noise ratio.
There are a variety of blocking buffers, not one of which is ideal for every combination of plate type, assay format, and detection system. Every blocking buffer represents a compromise between reducing background and maintaining specificity. Use this guide to help decide which type of blocking buffer is best suited for your specific application.
Detergent-based blocking buffers
Non-ionic detergents, such as tween-20, provide a convenient inexpensive blocking solution. However, detergents are not recommended as the sole blocking method: detergents are temporary blockers since they can be stripped by washing with water or aqueous buffer. Detergents are primarily useful as a secondary blocking agent; when included in the wash buffer, detergents can actively block sites on the plate surface that become exposed as weakly associated proteins are washed away.
| Advantages of non-ionic detergents | Disadvantages of non-ionic detergents |
|---|---|
| Inexpensive, despite higher concentration requirements | ineffective as a sole blocking method |
| highly stable, able to be stored as working solutions at room temperature | may cause the dissociation of molecules bound by noncovalent interactions |
| Increase the effectiveness of washes by encouraging the dissociation of weakly bound molecules and blocking the resulting exposed binding sites | may interfere with HRP detection systems |
| incompatible with lipopolysaccharides due to their ability to outcompete these molecules |
Protein Blockers
Protein blockers are a permanent blocking solution, and plates only need to be treated once for effective blocking. Protein blockers can also be added to the diluents used in subsequent steps to further reduce background signal. The most common blocking proteins include: bovine serum albumin (BSA), nonfat dry milk, and whole normal serum. Each has its own set of advantages and disadvantages:
| BSA | Nonfat dry milk | Whole normal serum | |
|---|---|---|---|
| Advantages | Inexpensive | Inexpensive | Effective at blocking all nonspecific interactions, including protein-protein interactions |
| Effective at concentrations as low as 1-3% | Effective at concentrations as low as 0.1-0.5% | Acts as protein stabilizer | |
| Well documented efficacy | Highly stable in dry form | ||
| Compatible with protein A | More effective at blocking covalent interactions | ||
| Disadvantages | High lot-to-lot variability due to variable fatty acid content | May cross-react with phospho-specific antibodies | Cross-reacts with protein A and anti-IgG antibodies |
| May cross-react with some classes of antibodies | Incompatible with alkaline phosphatase | Expensive | |
| Less effective at blocking covalent interactions | May cause overall higher background | Requires up to 10% concentration |
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