FITC Conjugated Antibody

What is the FITC conjugated antibody?

FITC-Conjugated Antibody denotes an antibody that is chemically labeled with FITC (Fluorescein Isothiocyanate), a fluorescent dye frequently employed for tagging proteins, peptides, or antibodies. This conjugation enables the antibody to function as a fluorescent marker in several biological experiments, especially in immunofluorescence, flow cytometry, and confocal imaging. FITC absorbs light at 495 nm in the blue spectrum and emits light at around 519 nm in the green spectrum, rendering it an efficient tool for visualizing the localization and expression of target proteins.

Preparation FITC-Conjugated Antibodies

The conjugation of FITC to antibodies entails a covalent interaction between the isothiocyanate group of FITC and the main amines located on the lysine residues of the antibody. This chemical conjugation establishes a stable thiourea bond, guaranteeing that the fluorescent label is securely affixed without impairing the antibody's capacity to bind to its specific antigen. FITC conjugation is typically conducted at mild circumstances to maintain the functional integrity of the antibody, since any structural or conformational alterations may compromise its antigen-binding capabilities.

Common fluorescent molecules

• Fluorescein isothiocyanate (FITC)
• Phycoerythrin
• Allophycocyanin
• Alexa Fluor: Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647
• Cyanine: Cy3, Cy5
• Dylight: Dylight 488, Dylight 550, Dylight 650
• Peridinin-Chlorophyll-Protein

Antibody conjugation technology at BOC Sciences

Applications of FITC-Conjugated Antibodies

Immunofluorescence: FITC-conjugated antibodies are extensively utilized in immunofluorescence microscopy to identify the presence and localization of antigens inside cellular or tissue specimens. The fluorescence released by FITC facilitates the imaging of individual proteins using a fluorescence microscope, so permitting a thorough examination of cellular structures and processes.

Flow cytometry: FITC-conjugated antibodies are utilized to mark cell surface or intracellular antigens. Cells tagged with FITC produce fluorescence upon laser excitation, facilitating the quantification and identification of various cell types according to their antigen expression.

Double immunofluorescence with FITC-conjugated antibody for desmin (green). (Scalia F., et al., 2022)

Confocal microscopy: FITC-conjugated antibodies are employed in confocal microscopy to get high-resolution, three-dimensional pictures of proteins within cells. This application is especially beneficial for examining protein distribution and interactions inside intricate biological structures.

Immunoassays: In several immunoassays, including ELISA, FITC-labeled antibodies facilitate the sensitive detection of antigens, providing both quantitative and qualitative information on protein presence and concentration.

Examples of FITC-conjugated antibodies

1. The FITC-conjugated anti-CD4 antibody specifically targets CD4 glycoproteins on T helper cells and is essential in immunology and HIV research. CD4 serves as a vital receptor for HIV entrance into cells, rendering this antibody essential for assessing immune function and tracking the advancement of HIV infection. The use of FITC-labeled anti-CD4 antibodies in flow cytometry has facilitated accurate measurement of T helper cell populations, an essential component in immune system evaluations and disease surveillance.
2. FITC-conjugated anti-CD8 antibodies, which identify CD8 proteins on cytotoxic T lymphocytes (CTLs), are essential for research on cellular immune responses, especially in cancer immunotherapy. The capacity to see and assess CTL activity via fluorescence-based approaches enhances comprehension of tumor immunity and the effectiveness of T cell treatments.
3. FITC-conjugated anti-CD19 antibodies are used to examine B cells, given that CD19 is a crucial indication of B cell growth and functionality. This antibody is particularly significant in the study of hematological malignancies, including leukemia and lymphoma, characterized by frequent B cell abnormalities. FITC-conjugated anti-CD19 antibodies are extensively utilized in flow cytometry for immunophenotyping, facilitating the characterization of B cell populations and the assessment of their functions in both normal immune responses and pathological conditions.
4. Because CD45 is a pan-leukocyte marker that is expressed on all nucleated hematopoietic cells, FITC-conjugated anti-CD45 antibody is an important tool that may be utilized. Several immunological tests may be performed using this antibody, which enables the differentiation of leukocytes from other cell types. Due to its usefulness in immunological profiling, it has become a vital tool in clinical diagnostics, particularly for conditions that are associated with dysfunctional immune cells, as well as for monitoring leukocyte populations in their healthy and sick forms.
5. FITC-conjugated anti-HLA-DR antibody is employed in immunological research to examine antigen-presenting cells, such as dendritic cells, macrophages, and B cells. It is crucial in transplant immunology and autoimmune disease studies.
6. The FITC-conjugated anti-α-tubulin antibody is a powerful tool widely used in cell biology and molecular research to visualize and study the structural organization of the cytoskeleton. Tubulin, a globular protein, forms microtubules—essential components of the cytoskeleton that play crucial roles in maintaining cell shape, enabling intracellular transport, and facilitating cell division. The α-tubulin subunit, along with β-tubulin, polymerizes to form the microtubule filaments, making it a significant marker for studying cellular architecture, mitosis, and cell dynamics.

Delivery of an FITC-conjugated anti-α-tubulin antibody into HepG2 cells. (Sabharwal P., et al., 2020)

Advantages of FITC-conjugated antibodies

The great sensitivity and adaptability of FITC-conjugated antibodies has been identified as one of the most significant benefits of these antibodies. The intense fluorescence that is produced by FITC makes it possible to detect proteins that are present in tiny quantities. This is especially useful in tests that need exact quantification. Furthermore, FITC-conjugated antibodies may be utilized in a broad variety of experimental formats, which makes them a flexible instrument that can be utilized for both in vitro and in vivo research. Researchers are able to monitor dynamic biological processes within living cells because to the capacity of FITC to release intense fluorescence upon activation. This property also enables real-time imaging.

Limitations of FITC-Conjugated Antibodies

Antibodies that have been conjugated with FITC have a number of intrinsic limitations, despite the fact that they are widely used. The process known as photobleaching, in which the fluorescent signal decays after being exposed to light for an extended period of time, is a significant disadvantage that restricts the amount of time that imaging investigations may be conducted. The fluorescence intensity of FITC is known to diminish in acidic settings, which might impair the dependability of results acquired from experiments conducted in such conditions. pH sensitivity is another issue that has to be addressed before it can be considered. In response to these limitations, newer fluorescent dyes that have higher photostability and pH resistance are being developed. However, FITC continues to be a mainstay in many laboratories due to the fact that it is both cost-effective and easy to use.