Sandwich Fluorescent Assay

Sandwich Fluorescent Assay is a type of immunoassay designed for the quantitative detection of target analytes in a sample. It utilizes a "sandwich" format where the target analyte is captured and detected using two specific antibodies and a fluorescent signal. This assay is valued for its high specificity and sensitivity in detecting a wide range of biological and chemical substances.

Technical Content

Principle of Operation

• Sandwich Format: The assay uses a pair of antibodies (or other specific capture and detection molecules) that bind to different epitopes of the target analyte. One antibody is immobilized on a solid support (e.g., a microplate), and the other is labeled with a fluorescent tag.
• Fluorescence Detection: The target analyte is "sandwiched" between these two antibodies. The presence of the target is detected by measuring the fluorescence emitted by the labeled antibody after it binds to the target.

Components

• Capture Antibody: An antibody (or another binding molecule) specific to the target analyte, immobilized on a solid support such as a microplate or bead.
• Target Analyte: The substance whose concentration is being measured, present in the sample.
• Detection Antibody: An antibody labeled with a fluorescent dye that binds to a different epitope of the target analyte, forming a "sandwich" complex.
• Detection System: Equipment such as a fluorescence microplate reader or fluorometer used to measure the fluorescence emitted by the labeled detection antibody.

Procedure

• Coating: The capture antibody is coated onto a solid support (e.g., wells of a microplate) and allowed to bind.
• Blocking: The support is treated with a blocking buffer to prevent non-specific binding.
• Sample Addition: The sample containing the target analyte is added to the wells. The target analyte binds to the capture antibody.
• Washing: Unbound components are removed through washing steps.

Sensitivity and Specificity

• Sensitivity: The assay is highly sensitive and can detect low concentrations of target analytes due to the amplification of the signal by the fluorescent detection system.
• Specificity: The assay is designed to minimize cross-reactivity and accurately detect the target analyte due to the use of two specific antibodies.

Applications

• Clinical Diagnostics: Measurement of biomarkers, proteins, or hormones in patient samples.
• Research: Quantification of proteins, nucleic acids, or other molecules in biological research.
• Environmental and Food Testing: Detection of contaminants or specific substances in environmental and food samples.

Advantages

• High Sensitivity and Specificity: Allows for accurate and precise measurement of target analytes even at low concentrations.
• Quantitative Results: Provides quantitative data on analyte concentration.
• Versatility: Suitable for a wide range of targets and applications.