Fluorescent Dyes for Antibody Labeling

Fluorescent antibody labeling technology uses chemical methods to covalently couple fluorescent dyes to specific antibodies. After the antibody-fluorescent complex specifically binds to the corresponding antigen, the fluorescent signal is observed through instruments such as fluorescence microscopes to achieve the positioning, qualitative or quantitative detection of the target sample. It provides researchers with an effective method to study molecular and cellular structures in biological systems.

What is Fluorescent Antibody?

Fluorescent antibodies are a commonly used biotechnology tool that combines antibody molecules with fluorescent dyes to detect, localize or quantify specific antigens in biological systems. This labeling technique relies on the high specificity of antibodies for their target antigens, which are usually proteins, carbohydrates or other macromolecules. The main feature of fluorescent antibodies is that they are conjugated to fluorescent molecules, which are chemically linked to the antibodies. When fluorescently labeled antibodies bind to their target antigens, the fluorescent molecules emit a visible light signal when exposed to light of a specific wavelength. This enables researchers to observe and analyze the presence and distribution of the target antigen using a microscope or other detection equipment. Today, fluorescent antibodies are widely used in a variety of research applications, including analyzing the distribution of proteins within cells, observing antigen expression in tissue sections, and detecting cell surface markers in flow cytometry.

Fig. 1. Fluorescent labeled antibodies.

Fluorescent Antibody Labeling Protocols

Immunofluorescence technology is a widely used method in cell and histological research for observing the localization and expression levels of specific proteins or molecules in biological samples. The fundamental principle involves the use of immunological techniques, where specially designed antibodies bind to the target proteins or molecules, followed by visualization through secondary antibodies labeled with fluorescent dyes. Using a fluorescence microscope, the position and expression levels of the target proteins in the sample can be immediately observed. Currently, based on different detection principles, immunofluorescence technology is typically divided into two methods: direct immunofluorescence assay and indirect immunofluorescence assay.

• Direct Fluorescent Antibody

The direct immunofluorescence assay (DFA) uses a fluorescently labeled antibody to directly bind to the antigen present in the test sample, allowing for the detection of the antigen. This method is simple to operate and highly specific, but it has relatively low sensitivity. It requires a specific labeled antibody for each antigen being detected. Currently, DFA can be used for immunofluorescence examinations in tissue pathology (such as kidney biopsy, skin biopsy, and lupus band tests), as well as for detecting pathogen antigens.

• Indirect Fluorescent Antibody

The indirect immunofluorescence assay (IFA) involves the reaction of the antigen in a substrate with the antibody present in the test sample, followed by the addition of a fluorescently labeled secondary antibody to detect the presence of the antibody in the sample. This method is often used to detect unknown antibodies in the sample and has a sensitivity that is 5 to 10 times higher than the direct method. Currently, IFA is the most commonly used immunofluorescence technique for clinical detection of autoantibodies.

Fluorescent Antibody Labeling Methods

The core principle of fluorescent antibody labeling is based on the high-affinity binding between antibodies and their target antigens. Antibodies can specifically recognize and bind to particular antigens, while fluorescent dyes are covalently coupled to antibody molecules, becoming an integral part of them. When excitation light is applied to the fluorescent dye, it transitions from the ground state to an excited state and then emits fluorescence at a specific wavelength. This fluorescence signal can be captured using specific optical filters and equipment, allowing for the labeling and localization of the target antigen. Generally, the fluorescent antibody labeling process involves two key steps: the binding procedure and quality control.

• Fluorescent Labeling Steps

1. Dilute the antibody to a concentration of 1-5 mg/ml using a sodium bicarbonate buffer (pH 9.8), or dialyze the antibody against the buffer to ensure that the lysine residues do not dissociate (maintaining their positive charge), while keeping most of the protein undenatured.
2. Place the dialysis bag into a 100 ml beaker containing freshly prepared 0.1 mg/ml sodium bicarbonate buffer (pH 9.8) and cover the beaker with aluminum foil to protect it from light. Stir at 4 °C overnight.
3. Dialyze the above antibody solution against PBS (phosphate-buffered saline) at 4 °C to terminate the reaction. Change the PBS at least three times until the absorbance at 480 nm is zero.
4. Add 0.5 g/L sodium azide to the solution. Afterward, store the conjugate at 4 °C in the dark, or aliquot and freeze it at -20 °C for long-term storage.

• Fluorescent Antibody Test

The conjugation rate can be determined using standard immunofluorescence staining assays, or the labeling quality can be evaluated by measuring the F/P ratio.

• Take 0.2 ml of the conjugate solution and add 2.8 ml of PBS (or use half the volume for both).
• Measure the absorbance at A490/A280, and use the FITC calibration curve to determine its concentration in µg/ml. Based on the extinction coefficient for IgG (approximately A280 = 1.2 for 1 mg/ml), calculate the F/P ratio, which represents the amount of FITC conjugated per mg of antibody. This allows for the assessment and comparison of the labeling quality across different batches.

Fluorescent Dyes for Antibody Labeling

Fluorescent dyes used for antibody labeling should meet the following requirements: they must have chemical groups that can form covalent bonds with protein molecules, ensuring the binding is stable and the unbound dye and its degradation products are easily removed. The dyes should also exhibit high fluorescence efficiency, retaining their fluorescence after binding to the protein. Additionally, the fluorescence color should contrast clearly with the background tissue, and the binding of the dye to the protein should not affect the protein's original biochemical or immunological properties.

• Fluorescein Dyes

The most widely used dye is fluorescein isothiocyanate (FITC), with an excitation wavelength of 488 nm and a maximum emission wavelength of 525 nm. FITC can bind to various antibody proteins and exhibits stable blue-green fluorescence in alkaline solutions. FITC-labeled antibodies are suitable for flow cytometry using 488 nm argon ion lasers and can be detected in the FL1 channel. It can also be used in fluorescence microscopy. However, the fluorescence intensity of FITC is sensitive to pH; it decreases as the pH value lowers.

• Cyanine Dyes

For small molecule fluorescent dyes, the Cy series includes Cy3, which has an excitation wavelength of 488 nm and a maximum emission wavelength of 570 nm. Cy3-labeled antibodies are suitable for flow cytometry with 488 nm argon ion lasers, detected in the FL2 channel, and are ideal for applications requiring small molecule dyes. However, Cy3 has lower fluorescence intensity than PE and is also suitable for fluorescence microscopy. Cy5, with an excitation wavelength of 633/635 nm and a maximum emission wavelength of 670 nm, is another small molecule dye ideal for flow cytometry with 633 nm argon ion lasers, detectable in the FL4 channel. Cy5 has lower fluorescence intensity than APC, and tends to bind non-specifically to monocytes and granulocytes, leading to potential false-positive results, although it is also suitable for fluorescence microscopy.

• Alexa Dyes

Excitation and emission spectra for the Alexa dyes range from all the visible light wavelengths to some infrared ones, so they can be used for a variety of applications. Alexa Fluor 488 (488 nm excitation wavelength, 519 nm maximum emission wavelength) is used widely in flow cytometry using 488 nm argon ion lasers on the FL1 channel. It has great photostability and works very well for fluorescence microscopy and can remain stable across a broad pH range (pH 4-10). Alexa Fluor 488 has been a favorite over the older fluorescent dyes like FITC and Cy2 because it is highly stable and reliable.

• Protein Dyes

Among the protein dyes, there is Phycoerythrin (PE) with a wavelength of 488 nm excitation and 575 nm emission. PE antibodies can be fluoresced using 488 nm argon ion lasers for flow cytometry and detected in the FL2 channel. PE is also strong fluorescence quenching, so not good for standard fluorescence microscopy, but great for laser confocal microscopy. A different protein dye, Allophycocyanin (APC), is excitation wavelength 633/635 nm and maximum emission wavelength 660 nm, and is compatible with flow cytometry using helium-neon lasers or red diode lasers. PreCP is a Peridinin-Chlorophyll Protein (PreCP), an excitation wavelength of 488 nm and an emission wavelength of 677 nm that can be used in conjunction with either FITC or PE, and has less spectral overlap and less non-specific binding to myeloid cells, making it best suited for high-expression target detection.

• PE Conjugated Composite Dyes

PE is a common dye derived from red algae that emits orange-yellow fluorescence after laser excitation, with a maximum emission wavelength of 575 nm. PE has excellent absorbance and high quantum yield, making it suitable for labeling proteins with low expression levels. For example, PE-Cy5 is a composite dye, where the excitation energy is transferred from PE to Cy5, with a maximum emission wavelength of 667 nm. Due to minimal spectral overlap between PE-Cy5 and FITC or PE, there is less fluorescence interference. PE-Cy5 is often used in combination with FITC or PE in experiments.

What is Fluorescent Antibody Used For?

Fluorescently labeled antibodies can be used in a wide range of applications due to their high sensitivity, specificity, and ability to enable multiplexing, making them valuable in fields such as cell biology, flow cytometry, clinical diagnostics, gene expression, and protein interaction studies.

• Cell Imaging

Modern cell biology depends on cell imaging to track the movement and dynamics of molecules, proteins and organelles inside cells. Antibodies are fluorescently labeled so they stick to antigens on the cell surface or within the cell and can then be mapped and measured by measuring the amount of target molecules through fluorescence signal under the microscope. Specifically, multiplexed fluorescence labelling means that scientists can scan multiple targets at once and thus get more functional information about the cell. Combining fluorescently labelled antibodies and fluorescence microscopy, researchers can observe protein changes in real time – molecular movements, distribution, interactions. This is especially relevant to biological interactions such as cell signal transduction, receptor internalisation and protein-protein interactions. What's more, fluorescently labeled antibodies also allow precise localisation of particular proteins inside the cell, such as in the nucleus, mitochondria and endoplasmic reticulum, in order to expose their function and regulatory activity.

• Flow Cytometry

Flow Cytometry is a widely employed method for the fast sorting and quantification of huge numbers of cells. Antibodies that are fluorescently labeled will bind different antigens to the surface of the cell, and flow cytometry uses multiplexed fluorescence signals to locate different markers on each cell. This lets scientists assess cell types, function and states simultaneously. For instance, in immunology, fluorescent antibodies can be used to identify different immune cell populations, so that the activity of their immune response can be measured. Target cells can be labelled with these antibodies too, and filtered cells can be isolated from samples via the cell sorting capabilities of flow cytometry. By labelling tumour antigens with fluorescent antibodies in tumor immunology, we can dissect cancer cells and further examine their biology.

• Immunohistochemistry

Immunohistochemistry is a technique that uses antibodies to bind to target molecules in tissue sections and amplify their presence through colorimetric or fluorescent signals, allowing the localization and distribution of molecules in tissues to be studied. The use of fluorescently labeled antibodies in IHC allows for the detection of the location and expression levels of molecules in tissue sections, providing sensitive and highly specific signals, especially in the detection of tumor markers, neurological research, and other disease biomarkers. By using different fluorescent dyes to label multiple antibodies, multiplex analysis can be performed on the same tissue section, enabling the simultaneous detection of several targets and their interactions within the tissue. For example, in tumor microenvironment studies, fluorescently labeled antibodies can reveal interactions between tumor cells and immune cells or stromal cells.

• Clinical Diagnostics

Fluorescently labeled antibodies are also used extensively in clinical diagnosis, for example, in pathology and infectious disease. Fluorescence microscopy lets doctors see the underlying pathology on sections of tissue and identify types and stages of disease. Fluorescent antibodies are used in cancer diagnosis to see tumour markers that tell doctors about tumour grade and outcome. In infectious disease diagnostics, such antibodies can rapidly recognise bacteria. When testing for bacterial or viral infections, for example, antibodies specific for fluorescent labelling will directly show the pathogen in patient fluids to accelerate diagnosis and speed up clinical treatment.

• Gene Expression and Protein Interaction Studies

Antibodies fluorescently labeled by fluorescent tags are used in gene expression and protein-protein interaction studies. They can be used in gene expression research to see what amount of a given protein and where it is located within the cell, which is invaluable information to know about gene regulation. And by using fluorescence confocal microscopy and fluorescent antibodies, it's possible to track protein-protein interactions. When they label two antibodies together, for instance, they can see how the target proteins co-localise inside the cell and discover their biological roles and interactions.

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