Immunoassays such as immunohistochemistry (IHC), immunofluorescence (IF), ELISA, and Western blotting are the techniques used in research to detect and quantify biomolecules through precise binding of antibodies with target proteins. However, non-specific binding can generate background noise, obscure true signals, making experimental results less reliable.
Researchers use blocking agents to address this challenge. These are special substances that occupy non-specific binding sites before antibody application, which helps enhance specificity, accuracy, and reproducibility of immunoassays. As a result, researchers can obtain clean, interpretable, and reproducible data.
Understanding Non-Specific Binding
Antibodies bind to unintended targets due to several mechanisms including:
- Electrostatic forces
- Hydrophobic interactions
- Binding to Fc receptors
These non-specific interactions in samples lead to staining of areas that do not contain the target antigen.
This produces background noise, resulting in inaccurate data and also making experiments less reproducible. The use of blocking agents is the most effective strategy to address this challenge.
Primary Role of Blocking Agents
Blocking agents occupy sites that should not interact with antibodies. These sites may exist on:
- Tissue sections
- Cell surfaces
- Assay plate surfaces
Preventing non-specific binding helps in improving the signal-to-noise ratio. Blocking agents reduce noise and help improve signal quality. Researchers can now easily distinguish the intended antibody interaction with the target from random interactions.
This helps visualize protein localization in tissue sections in qualitative analysis and precise measurement of protein levels in quantitative assays. Blocking agents contribute to experimental reproducibility.
With pre-treatment of samples, researchers can achieve consistent results across multiple experiments and different laboratories.
So, blocking agents can help create a controlled environment, thereby improving the accuracy, reliability, and interpretability of immunoassay results.
Types of Blocking Agents
Different forms of blocking agents are used in different experimental conditions. The choice of blocking agents depends on:
- Type of immunoassay
- The nature of the sample
- The antibodies being used
The following are the categories of blocking agents:
Serum-based Blockers
Derived from the serum of non-immunized animals, serum-based blockers contain a complex mixture of proteins that effectively occupy non-specific binding sites. Their diverse protein composition allows them to block a wide range of unwanted interactions, including Fc receptor binding and non-specific antibody attachment.
It is important to ensure that the species is compatible with the secondary antibody. For example, when secondary antibodies are derived from goats, normal goat serum should be used as the blocking agent. Matching species helps prevent cross-reactivity and reduce background staining by ensuring that the secondary antibody does not bind non-specifically to endogenous proteins or immunoglobulins present in the sample.
Protein-based Blockers
Protein-based blockers consist of purified proteins that are used to saturate non-specific binding sites in immunoassays. Commonly used protein-based blockers include bovine serum albumin (BSA), casein, and gelatin. Their defined composition and consistency help reduce variability between experiments and improve reproducibility.
These blockers are used in assays where controlled experimental conditions are essential. They are effective at preventing non-specific adsorption of antibodies to assay plates or membranes, thereby improving signal clarity. However, protein-based blockers contain a limited range of proteins compared to serum-based blockers, making them less effective in blocking Fc receptor-mediated interactions in tissue samples.
Synthetic or Commercial Blockers
These are chemically defined or polymer-based formulations. Synthetic or commercial blockers are typically supplied as ready-to-use solutions. This ensures consistency and reduces preparation time. Their defined composition also minimizes batch-to-batch variability, making them suitable for standardised and high-throughput applications.
These blockers are especially useful in ELISA and Western blotting. These assays essentially require reproducibility and ease of use. However, they may require optimization for complex tissue samples, as their blocking efficiency can vary depending on antibody properties and assay conditions.
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