Bioconjugation in Fluorescence Immunoassay (FIA)
What is fluorescence immunoassay?
Fluorescence immunoassay (FIA) is based on the principle of antigen-antibody reaction, first fluorescein is conjugated to a known antigen or antibody, and then this fluorescent antibody (or antigen) is used as a probe to detect the corresponding antigen (or antibody) in serum, body fluid, cells or tissues. The antigen-antibody complex formed in the tissue or cell contains labeled fluorescein. Fluorescein is irradiated by external excitation light, and in a very short time (10-8 ~ 10-9 s), excited molecules are generated, and visible light with a longer wavelength than the excitation light is released. The antigen or antibody to be detected can be qualitatively and quantitatively analyzed through indicators such as fluorescence intensity and expression position. Common fluorescence immunoassays include flow cytometry, time-resolved immunofluorescence assay, fluorescence polarization immunoassay and fluorescence enzyme immunoassay.
Time-resolved fluorescence immunoassay (TRFIA): A non-radioactive microassay technique that labels an antigen or antibody with lanthanide elements and is combined with time-resolved techniques.
Fluorescence polarization immunoassay: Using the principle of antigen-antibody reaction, the content of small molecules in body fluids is determined according to the difference of fluorescence polarization degree between fluorescein labeled antigen and antigen-antibody complex.
Fluorescein immunoassay: The enzyme-labeled antigen (antibody) is used to react with the antigen (antibody) to be tested, and the fluorescent substrate is reacted with enzyme to generate a stable and efficient fluorescent substance through enzymatic reaction, and the content of the antigen or antibody to be tested is determined by measuring the fluorescence intensity.
Flow cytometry: By binding individual cells to specific antibodies labeled with different fluorescent dyes, multi-parameter quantitative analysis of cells, including cell surface markers, intracellular protein expression, cell cycle, DNA content, etc.
Types of fluorescence immunoassay
Fluorescence immunoassay can be divided into direct method, indirect method, complement method and double labeling method.
Direct fluorescence immunoassay: In the direct fluorescence immunoassay method, a fluorescently labeled antibody is applied directly to the sample containing the target antigen. This approach allows for immediate visualization of the antigen-antibody complex, with the emitted fluorescence directly correlating to the presence of the target molecule. The simplicity and rapid execution of this method make it suitable for straightforward applications, particularly when dealing with well-characterized antigens. However, the sensitivity may be limited, as the direct approach may not fully capitalize on amplification strategies.
Indirect method: Also known as double antibody method. Using fluorescently labeled antibodies to identify unknown antigens or unknown antibodies, fluorescently labeled anti-globulin antibodies can detect a variety of unknown antigens or antibodies, its sensitivity is 5 to 10 times higher than the direct method, the disadvantage is that there are more components to participate in the reaction, the steps are complicated, and the need for more controls. It is often used for the detection of various autoantibodies.
Complement method: Anti-complement antibodies are labeled with fluorescein to identify unknown antigens or antibodies. This method requires only one labeled anti-complement antibody. Because complement binding to the antigen-antibody complex is not species-specific and is not limited by the type of animal with known antibodies or serum to be tested, a variety of antigen-antibody systems can be detected. The disadvantage is that it is easy to produce non-specific interference, requires more control, and is not easy to store for a long time due to the instability of the complement.
Double labeling method: The same substrate sample is detected by labeling different antibodies with fluorescein isothiocyanate (FITC) and tetraethylrhodamine (RB200), and if there are two corresponding antigens, the fluorescence of different colors is displayed.
Bioconjugation in fluorescence immunoassay
Bioconjugation is a fundamental process in fluorescence immunoassays, playing a crucial role in linking biomolecules, such as antibodies, to fluorescent labels. This chemical coupling enhances the assay's overall performance by increasing the visibility and stability of the fluorescent signal. The success of FIA hinges on the careful selection of bioconjugates, which must maintain their biochemical properties while providing robust fluorescence.
The process of bioconjugation typically involves creating stable covalent bonds between fluorescent dyes and biomolecules. This ensures that the fluorescent label remains attached during the assay, enabling reliable detection of the target analyte. Various reagents are utilized for this purpose, including N-hydroxysuccinimide (NHS) esters, which react with amino groups on proteins, and maleimide derivatives that target thiol groups. These reactions facilitate the formation of stable conjugates, which are essential for achieving consistent and reproducible results.
In addition to covalent bonding, bioconjugation can also be achieved through non-covalent interactions. Techniques such as avidin-biotin binding leverage the high affinity between biotin and avidin, allowing for effective labeling without altering the biomolecule's native structure. This method is particularly advantageous when the preservation of the protein's functionality is paramount, as it minimizes the risk of denaturation.
Furthermore, the advent of click chemistry has revolutionized bioconjugation in fluorescence immunoassays. This innovative approach utilizes bioorthogonal reactions that occur selectively in living systems, allowing for efficient and specific labeling of biomolecules. Click chemistry minimizes side reactions and enhances the specificity of the labeling process, making it an attractive option for developing advanced fluorescent probes.
Site-specific labeling is another key aspect of bioconjugation, enabling targeted attachment of fluorescent labels to particular amino acids within a protein. This level of precision enhances the performance of fluorescence immunoassays by reducing background noise and improving signal clarity. By ensuring that the fluorescent tag does not interfere with the antibody's binding sites, researchers can achieve higher sensitivity and specificity in their assays.
Common bioconjugates used in fluorescence immunoassay
Antibody-dye conjugates: Fluorescent dyes, such as fluorescein isothiocyanate (FITC) and rhodamine, are covalently linked to antibodies, enabling direct visualization of the antigen-antibody interaction. These dyes are chosen for their brightness and photostability, which enhance signal detection.
Avidin-biotin systems: Bioconjugates involving biotinylated antibodies combined with streptavidin-dye conjugates leverage the strong affinity between avidin (or streptavidin) and biotin. This system allows for the amplification of the fluorescent signal, as multiple streptavidin molecules can bind to a single biotinylated antibody. The versatility of this approach makes it suitable for various applications, including the detection of low-abundance targets.
Phycobiliprotein conjugates: Phycobiliproteins, such as allophycocyanin (APC), are also utilized as fluorescent labels due to their exceptional brightness and narrow emission spectra. When conjugated to antibodies, these proteins facilitate sensitive detection in multiplex assays, where distinguishing between multiple targets is essential.
Lanthanide chelates: Lanthanide chelates are notable for their long fluorescence lifetimes and high molar absorptivity. These characteristics make them ideal for time-resolved fluorescence assays, where background noise can be minimized. The use of lanthanide chelates allows for highly sensitive detection of antigens, particularly in complex samples.
Quantum dot-antibody conjugates: Quantum dots are semiconductor nanocrystals that offer size-tunable fluorescence and remarkable photostability. Their unique optical properties make them valuable in fluorescence immunoassays, particularly for multiplexing applications, where several targets are detected simultaneously. When conjugated to antibodies, quantum dots enable high-resolution imaging and enhanced sensitivity.
Probes for specific applications: Fluorophore-labeled oligonucleotide beacons or peptide probes are employed in fluorescence immunoassays. These probes can act as quenchers and are designed for specific binding interactions, enhancing the specificity and sensitivity of the assay.
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Application of bioconjugation in fluorescence immunoassay
FIA is a highly sensitive and specific assay that is widely used in the biomedical field, especially in the determination of cytokines, microorganisms and hormones. By using fluorescently labeled antibodies or antigens, FIA enables rapid and accurate detection of target molecules.
Cytokine assay
Cytokines not only participate in the immune response, but also play an important role in the occurrence and development of tumors and inflammation. Therefore, the accurate determination of cytokine content is very important to understand its pathophysiological effects. FIA often uses fluorescent-labeled antibodies that bind to cytokines, such as fluorescein-labeled monoclonal antibodies, to detect cytokines such as tumor necrosis factor α (TNF-α).
Microbiological diagnosis
Using the biotin-Streptavidin system, it is possible to bind antibodies labeled with fluorescent dyes such as fluorescein or Rhodamine to specific pathogens, enabling rapid detection of infectious pathogens such as influenza viruses and toxoplasma gondii. For example, the use of fluorescently labeled antibodies can simultaneously detect IgM and IgA antibodies against the influenza virus in a patient's serum, greatly reducing the diagnostic time.
Nucleic acid diagnosis
Europium-labeled streptavidin-biotin probes can be used for rapid detection of HIV RNA and DNA of other pathogens, showing high sensitivity and specificity. This method provides an effective means for early screening and monitoring of virus infection.
Hormone immunoassay
By using fluorescence-labeled beta-human chorionic gonadotropin (beta-hCG) antibodies, hormone levels can be detected in pregnant women's serum to help determine pregnancy status. In addition, detection of thyroid hormones is also achieved through FIA, and the use of fluorescently labeled antibodies (such as those labeled with Rhodamine) enables more accurate quantification of hormone levels in the serum.