Anti-FITC monoclonal antibody, clone 20G/3A4

Fluorescent probe technology with its high sensitivity and selectivity is widely used in chemical and biological experiments and analysis. It has increasingly replaced radioactive isotope labelling. Researchers often use fluorescein analogs, rhodamines and cyanines as fluorescent dyes. Among the fluorescein derivatives are dyes such as FITC and FAM. These dyes attach well to -OH, -SH, and -NH₂ which is great for fluorescent probe detection.

Today, the most common fluorescent dye is FITC. It consists of 389.4 moles and it appears as an orange-yellow crystal powder. Its dissolved forms in water and alcohol are yellow-green fluorescent and readily observed. The spectral nature of FITC is excitation wavelength of 494 nm and emission wavelength of 520 nm. Protein labeling and fluorescence microscopy in biomedical field are typically performed with FITC. The FITC-containing antibodies will be visible under the fluorescence microscope and will surface and target antigens in immunohistochemistry and immunofluorescence studies. FITC reacts with proteins' amino terminus and primary amines to create stable covalent bonds that permit protein fluorescence. This characteristic makes it widely applicable in antibody labeling, lectin labeling, and other similar uses. FITC is often used in fluorescence tracing to track the movement of molecules from one cell to the next in order for scientists to know what's happening inside. Furthermore, since polysaccharides lack chromophores and are therefore inaccessible to tools like fluorescence spectrophotometers, fluorescent labelling can be used to detect polysaccharides. FITC has been discovered to be a good polysaccharide labeller, and this fluorescein tracing technique is one that could be used to probe polysaccharide structure and metabolism in detail.

Figure 1. Representation of the chemical reaction forming FITC@Ch
(Source: Caprifico AE, et al. 2021)

The reaction between FITC and proteins primarily relies on the reactive isothiocyanate group (-N=C=S) in its chemical structure. This group can undergo a conjugation reaction with amino groups in proteins, particularly primary amines, resulting in stable fluorescent labeling. FITC mainly reacts with primary amines in proteins, such as lysine residues and N-terminal amino groups. This is because these amino groups can be deprotonated at physiological pH, forming reactive amines that readily react with the isothiocyanate group. The product of the reaction between FITC and amines is a stable thiourea structure, which provides long-lasting fluorescent properties in biological experiments.

Anti-Fluorescein Isothiocyanate monoclonal antibody