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Labeled Streptavidin-Biotin Technique: Principles and Applications

The Labeled Streptavidin-Biotin Technique, often referred to in English as the Labeled Streptavidin-Biotin Method or Labeled Streptavidin-Biotin System, is a cornerstone technology in molecular detection, immunoassays, and histochemistry. This article provides a comprehensive overview of its principle, key components, applications, and advantages to fully address your inquiries.

What is the Labeled Streptavidin-Biotin Technique?

It is an ultra-sensitive detection and amplification system based on the nearly irreversible non-covalent interaction between streptavidin (or avidin) and biotin (Vitamin B7). This binding is one of the strongest in nature (Kd ≈ 10⁻¹⁵ M), providing exceptional specificity and stability. In the “labeled” version, the streptavidin molecule itself is conjugated to a detectable marker (label), such as:

• Enzymes: Horseradish Peroxidase (HRP), Alkaline Phosphatase (AP)
• Fluorophores: FITC, PE, Alexa Fluor dyes
• Isotopes: Iodine-125 (¹²⁵I)
• Nanoparticles: Gold, quantum dots

This direct conjugation allows for straightforward and powerful detection of a target molecule.

How Does It Work? (The Principle)

The technique typically involves a multi-step process, with the most common format being the Labeled Streptavidin-Biotin (LSAB) method for immunohistochemistry (IHC) or ELISA:

1. Primary Antibody Incubation: A specific, unlabeled primary antibody binds to the target antigen.
2. Biotinylated Secondary Antibody Incubation: A secondary antibody, which is raised against the host species of the primary antibody and is conjugated to multiple biotin molecules, binds to the primary antibody.
3. Labeled Streptavidin Incubation: The streptavidin protein, which is conjugated to a label (e.g., HRP enzyme), is added. Each streptavidin tetramer can bind to multiple biotin molecules on the secondary antibody, creating a large complex and significantly amplifying the signal.

This cascade results in the accumulation of many label molecules at the site of the target antigen, enabling highly sensitive detection.

Key Advantages of the System

• Extreme Sensitivity: The signal amplification is superior to direct or two-step methods because multiple biotins can bind to a single secondary antibody, and each streptavidin can bind multiple biotins.
• High Specificity and Affinity: The strong biotin-streptavidin interaction minimizes non-specific binding and background noise.
• Versatility: The same biotinylated secondary antibody can be paired with a vast array of labeled streptavidin reagents (HRP, fluorescent, etc.), making the system adaptable for various applications without changing the initial steps.
• Stability: The complexes are very stable under a wide range of conditions (pH, temperature, denaturing agents).

Major Applications

This technique is a workhorse in biomedical research and diagnostics:

• Immunohistochemistry (IHC) & Immunofluorescence (IF): For visualizing protein distribution in tissue sections and cells.
• Enzyme-Linked Immunosorbent Assay (ELISA): For detecting soluble antigens or antibodies with high sensitivity.
• Western Blotting: For detecting specific proteins separated by gel electrophoresis.
• Flow Cytometry: For tagging and analyzing cell surface and intracellular markers.
• Nucleic Acid Detection (In-situ Hybridization, Blotting): Using biotin-labeled DNA/RNA probes followed by detection with labeled streptavidin.
• Affinity Purification: Using immobilized streptavidin beads to capture and purify biotin-tagged molecules like proteins or nucleic acids.

Common Variations and Related Terms

You may encounter these related terms, which are specific implementations of the core concept:

• LSAB (Labeled Streptavidin-Biotin) Method: Described above.
• ABC (Avidin-Biotin Complex) Method: Involves pre-forming a large complex between avidin/streptavidin and biotinylated enzyme before adding it to the biotinylated secondary antibody. This can offer even greater amplification.
• BAB (Bridged Avidin-Biotin) Method: A more complex three-step method.

In modern practice, streptavidin is strongly preferred over avidin due to its near-neutral isoelectric point, which results in much lower non-specific electrostatic binding to tissues and thus lower background noise.

Conclusion