标记生物素亲和素法英文翻译

The Labeled Biotin-Avidin Method is a powerful and widely used technique in molecular biology, immunology, and histochemistry for the detection of target molecules with极高的灵敏度 (extremely high sensitivity) and specificity. This system leverages the exceptionally strong non-covalent interaction between biotin (a small vitamin) and avidin or its bacterial counterpart, streptavidin (a large tetrameric protein).

The methodology typically involves three key steps:

1. Biotinylation: The molecule of interest (e.g., an antibody, nucleic acid probe, or other protein) is conjugated with biotin, forming a “biotin-labeled” probe.
2. Binding: The biotinylated probe binds specifically to its target (e.g., an antigen on a cell, a specific DNA sequence).
3. Detection: Avidin or streptavidin, which is itself conjugated to a detectable marker (e.g., an enzyme like HRP or AP, a fluorophore, or gold nanoparticles), is introduced. It binds rapidly and firmly to the biotin tags, allowing for visualization or quantification of the target.

Common systems derived from this method include the ABC (Avidin-Biotin Complex) method and the LSAB (Labeled StreptAvidin-Biotin) method.

A Comprehensive Guide to the Labeled Biotin-Avidin Method: Principles, Applications, and Protocols

The Labeled Biotin-Avidin Method is a cornerstone technique in laboratories worldwide. If you’re searching for this term, you likely have questions about how it works, why it’s so effective, and how to use it in your own research or diagnostics. This article aims to provide a complete overview, addressing the core needs behind this search query.

1. The “Why”: Unmatched Sensitivity and Signal Amplification

The primary reason researchers choose this method is its ability to significantly amplify the detection signal, leading to superior sensitivity compared to direct labeling methods.

• The Secret: The Biotin-Avidin Interaction

  • Biotin (Vitamin B7): A small molecule that can be easily attached to antibodies and other probes without significantly altering their biological activity.
  • Avidin/Streptavidin: A much larger protein. Each avidin or streptavidin molecule has four binding sites for biotin.
  • Strength: The binding affinity (Kd ≈ 10⁻¹⁵ M) is one of the strongest non-covalent interactions known in nature, making the bond virtually irreversible under most conditions.

• How Amplification Works:
  In a standard indirect detection method using a secondary antibody, you might get 3-5 enzyme molecules bound per primary antibody. In the biotin-avidin system, multiple biotin molecules are attached to a single primary antibody. A single streptavidin molecule (with 4 biotin-binding sites) can then bind to these multiple biotins. Furthermore, each streptavidin molecule can be pre-conjugated to multiple enzyme molecules (e.g., several HRP molecules), leading to an enormous accumulation of reporter molecules at the target site. This multi-layer amplification is the key to its high sensitivity.

2. The “How”: Key Techniques and Protocols

There are several established variations of the method, each with slightly different protocols:

• Labeled StreptAvidin-Biotin (LSAB) Method: The most common protocol.

  1. A biotinylated primary antibody binds to the target antigen.
  2. Enzyme-labeled Streptavidin (e.g., Streptavidin-HRP) is added, which binds to the biotin on the primary antibody.
  • Advantage: Simple, fast, and highly sensitive.

• Avidin-Biotin Complex (ABC) Method: An earlier, even more sensitive method.

  1. A biotinylated primary antibody binds to the target.
  2. A pre-formed complex of Avidin and Biotinylated Enzyme (e.g., biotinylated HRP) is added. This large ABC complex creates a massive lattice of enzyme molecules at the site of binding.
  • Advantage: Maximum signal amplification.
  • Disadvantage: More complex protocol, larger complex size may limit tissue penetration.

• Bridging Method: Used when a biotinylated primary antibody is not available.

  1. An unlabeled primary antibody binds to the target.
  2. A biotinylated secondary antibody (anti-host IgG) is added.
  3. Enzyme-labeled Streptavidin is added to detect the biotin.

3. The “Where”: Major Applications

This versatility makes the method applicable across numerous fields:

• Immunohistochemistry (IHC) & Immunocytochemistry (ICC): The dominant application. Used to visualize the distribution and localization of specific antigens (proteins) in tissue sections and cells with exceptional clarity.
• Enzyme-Linked Immunosorbent Assay (ELISA): Used as the detection system in sandwich ELISA and competitive ELISA formats to achieve very low detection limits for cytokines, hormones, and pathogens.
• Western Blotting: Detects low-abundance proteins on membranes by amplifying the signal from the primary antibody.
• Molecular Biology:
  • DNA/RNA Hybridization: Biotin-labeled nucleic acid probes are used to detect specific DNA or RNA sequences on blots (Southern, Northern) or in situ (FISH).
  • Flow Cytometry: Biotinylated antibodies are used in complex multi-color panels, followed by a common streptavidin-fluorophore conjugate, saving costs on directly labeled rare antibodies.
• Affinity Purification: Streptavidin conjugated to beads is used to capture and purify biotin-tagged molecules like proteins, nucleic acids, or even entire complexes (e.g., using BioID).

4. The “What”: Streptavidin vs. Avidin - A Critical Choice

While often used interchangeably, the choice between avidin and streptavidin is crucial.

• Avidin: Derived from egg whites. It has a high isoelectric point (pI ~10), meaning it is positively charged and can cause non-specific electrostatic binding to negatively charged cellular structures (e.g., nuclei), leading to background noise.
• Streptavidin: Derived from the bacterium Streptomyces avidinii. It has a near-neutral pI and is not glycosylated, resulting in significantly lower non-specific binding and much cleaner backgrounds. For virtually all modern applications, streptavidin is the preferred choice.

5. Advantages and Limitations

Advantages:

• Extremely High Sensitivity: Due to signal amplification.
• High Specificity: The biotin-streptavidin bond is very specific and strong.
• Versatility: Compatible with multiple detection platforms (chromogenic, fluorescent, chemiluminescent).
• Flexibility: The same streptavidin-conjugate can be used with any biotinylated primary antibody, reducing costs and simplifying reagent inventory.

Limitations:

• Endogenous Biotin: Some tissues (e.g., liver, kidney, breast) contain high levels of endogenous biotin, which can bind the streptavidin reagent and cause high background. This requires a blocking step with free avidin/streptavidin and subsequent biotin blocking.
• Size: The large size of the ABC complex can sometimes hinder penetration into dense tissue or complex cellular structures.
• Cost: The additional layers of reagents can increase cost compared to simple secondary antibody methods, though the gain in sensitivity often justifies it.

Conclusion