Antibody Dye Conjugation

What is the antibody dye conjugation?

Antibody dye conjugation is an essential method in molecular biology and biotechnology involving the chemical attachment of fluorescent dyes or other detection markers to antibodies. This procedure enables antibodies, which inherently bind to certain antigens with great affinity, to be utilized for diverse detection and imaging applications by conjugating them with a visible or quantifiable signal. Labeled antibodies are commonly utilized in many scientific, diagnostic, and therapeutic applications, especially in fluorescence microscopy, flow cytometry, and immunoassays. Antibody-dye conjugates are increasingly utilized in targeted therapeutics, such as antibody-drug conjugates (ADCs), where fluorescent labels facilitate real-time monitoring of drug distribution and effectiveness.

Study patients received infusion of antibody–dye conjugate. (Lu G., et al., 2020)

Preparation of Antibody-Dye Conjugates

Antibody dye conjugation denotes the chemical procedure of covalently linking dyes or other markers to antibodies. These dyes are often tiny organic compounds exhibiting fluorescent qualities, including fluorescein isothiocyanate (FITC), Alexa Fluor, and cyanine dyes (e.g., Cy3, Cy5), which emit light when excited. Conjugation often entails reactive groups present on both the dye and the antibody. Antibodies can be conjugated with various fluorescent dyes, such as amine-reactive and sulfhydryl-reactive dyes. Effortlessly synthesize molecular imaging probes by conjugating antibodies with fluorescent compounds. For instance, amine-reactive fluorescent dyes like FITC and sulfhydryl-reactive dyes such as fluorescein-5-maleimide specifically attach to the amino and sulfhydryl groups of antibody molecules, respectively. The latest work utilized N-hydroxysuccinimidyl ester (NHS) derivatives of the fluorescent dyes Alexa488 and ICG to mark the lysine residues of antibody molecules. The NHS derivatives, Alexa488-NHS ester and ICG-NHS ester, readily react with the amino groups of antibody molecules in a pH range of 7.5 to 9, resulting in fluorescence-labeled antibody molecules. The labeling efficacy of the dye to antibody may be regulated by the molar ratio of dye to antibody and the duration of the reaction. The labeling efficiency was 50% for Alexa 488-Herceptin and 89% for ICG-Herceptin conjugates.

Representation for the conjugation of Dye-NHS to antibody. (Tsuboi S., et al., 2020)

Antibody conjugation technology at BOC Sciences

Amplification effect of antibody dye conjugation

The fluorescence intensity is influenced not only by the quantity of antibody molecules or active antibody fragments but also by the ratio of fluorescent molecules to each antibody molecule. Typically, each antibody molecule may be attached with four to five fluorescent molecules with minimal impact on binding properties. As previously mentioned, additional parameters particular to the dye also contribute significantly. These encompass the excitation and emission wavelengths, quantum efficiency, extinction coefficient, and quenching rate. The direct conjugation of the main antibody to fluorescent or other markers offers several benefits. The likelihood of cross-reactivity diminishes when the specimen is treated with a single antibody. The cross-reactivity and nonspecific binding of the secondary antibody have been eradicated. The considerations about the binding avidity and affinity of the secondary antibody to the primary antibody are also disregarded. Ultimately, spatial resolution is optimized, resulting in more dependable measurements of semiquantitative elements.

To prevent the conjugation of the primary antibody to a fluorescent dye and the potential loss of antibody function or activity, a secondary antibody-fluorescent dye conjugate can be employed in a sandwich technique, wherein the antigen is bound to the primary antibody, which is in turn bound to a fluorochrome-conjugated secondary antibody that recognizes the primary antibody. The secondary antibody precisely interacts with the first antibody and may exhibit broader specificity. Secondary antibodies can be attached to several fluorescent dyes, providing many excitation/emission combinations. If the primary antibody is derived from a mouse, a secondary antibody from many other species, including rabbit, chicken, or goat, may be utilized as the secondary antibody. Typically, several secondary antibody molecules will attach to each original antibody molecule. Each second antibody molecule will be linked to four or five fluorescent molecules, therefore significantly enhancing the signal. In theory, according to the experimental scheme, there is also a third antibody, and the fourth antibody binds in order to play a signal amplification role.

The antibody-fluorescent dye conjugates. (Albrecht R M., et al., 2018)

Applications of antibody dye conjugation

Fluorescence Microscopy: In immunofluorescence microscopy, dye-conjugated antibodies are employed to identify specific proteins or structures within cells and tissues. By targeting specific antigens, these conjugated antibodies provide the visualization of the location and quantity of these proteins within cells, yielding crucial insights into cell biology, disease, and signaling pathways.

Flow cytometry: Flow cytometry utilizes fluorescently labeled antibodies to assess the expression of certain surface or intracellular markers in individual cells. Simultaneous use of numerous antibodies coupled to various dyes (multiplexing) facilitates the comprehensive characterization of cell populations in heterogeneous samples, including blood, malignancies, or immunological tissues.

Western blotting: In Western blot experiments, dye-conjugated secondary antibodies are frequently employed to identify target proteins that have been resolved by gel electrophoresis and subsequently transferred to membranes. The fluorescence generated by the attached antibody upon excitation offers a precise measurement of the presence and amount of the target protein.

Immunohistochemistry (IHC): Dye-conjugated antibodies are employed to stain tissue slices, enabling the viewing of antigen distribution and abundance within a tissue context. Fluorescent dye conjugation provides an alternative to chromogenic detection approaches, offering enhanced sensitivity and the capability for multiplexing.

ELISA (Enzyme-Linked Immunosorbent Assay): Fluorescent dye-conjugated antibodies or other markers are utilized in ELISA tests to measure the concentration of certain antigens or antibodies in a sample.

Benefits of antibody dye conjugation

High sensitivity: Fluorescent dyes possess large quantum yields, allowing them to generate intense fluorescence signals even at little concentrations. Conjugated antibodies have heightened sensitivity in identifying target antigens, particularly in tests characterized by low antigen prevalence.

Multiplexing capability: The use of several dyes with distinct emission spectra facilitates the concurrent examination of many targets inside a single sample, rendering it especially advantageous in intricate biological systems.

Real-Time tracking: In live-cell imaging applications, dye-labeled antibodies provide real-time monitoring of biological processes, offering dynamic insights into molecular interactions.

Non-radioactive: Fluorescent labeling provides a safer and more practical alternative to radioactive labeling, which necessitates specialized handling and disposal protocols.

Constraints of antibody-dye conjugation

Steric hindrance: The attachment of large dye molecules to an antibody can induce steric hindrance, potentially impairing the antibody's capacity to bind to its target antigen. Should the conjugated dye be positioned adjacent to the antigen-binding site (paratope), it may obstruct or diminish the antibody's capacity to engage with the antigen, resulting in attenuated or incomplete binding.

Suboptimal Dye-to-Antibody ratios: The degree of labeling (DOL), which refers to the quantity of dye molecules attached to an antibody, requires meticulous regulation. Insufficient dye molecules may yield insufficient signal detection, whereas an excess of dyes might cause quenching (diminution of fluorescence intensity) or compromise antibody functionality. Attaining the ideal dye-to-antibody ratio is essential for optimizing fluorescence intensity while maintaining antibody activity. Excessive conjugation may result in fluorescence self-quenching, whereas insufficient conjugation can diminish test sensitivity.

Fluorescence Quenching: Excessive attachment of dye molecules on the antibody may lead to fluorescence quenching, wherein the fluorescence signal diminishes or is extinguished due to interactions among proximate fluorophores. Quenching attenuates the fluorescence signal's intensity, hence diminishing detection sensitivity in tests such as flow cytometry or immunofluorescence.

Cost and Availability: Antibody-dye conjugates, particularly those of superior commercial quality, can be costly. Custom conjugation further increases research expenses, especially when specific dyes or high-purity antibodies are necessary. The expense of conjugation reagents and the intricacy of refining conjugation processes may restrict the practicality of employing antibody-dye conjugates in large-scale or high-throughput investigations.