Most Popular and Versatile Labels Conjugated to Antibodies for Detection of Antigen in Immunoassays

Guide to common labels conjugated to primary or secondary antibodies including biotin, FITC, and HRP.

Detection or visualization of the antigen of interest in immunoassays often requires labeling or conjugation of the antibodies with fluorophores, enzymes, or biotin. These labeling agents can be conjugated either to a primary antibody (direct assay) or to a secondary antibody (indirect assay). The direct assay uses a primary antibody specific for the antigen of interest directly bound to the conjugate for detection, and does not require the use of a secondary antibody. Whereas indirect assays use a primary antibody specific for the antigen of interest and a secondary antibody conjugated to the labeling agent for detection. The use of labeled secondary antibodies often amplifies the signal. While the use of conjugated primary antibodies increases specificity, reduces cross-reactivity and non-specific background caused by secondary antibody, and makes the assay faster.

Labels, such as biotin, fluorophores, or enzymes can be conjugated to primary or secondary antibodies via the primary amino groups in the lysine residues of the IgG antibody. Below are some of the most common labels typically conjugated to antibodies.

Biotin

Biotin is a small polypeptide often conjugated to antibodies, as it increases the signal and assay sensitivity. Three to six biotin molecules bind to the antibody and biotin has high affinity to avidin or streptavidin. The biotinylated antibody binds to streptavidin, forming an avidin-biotin complex, while streptavidin is conjugated to enzymes or fluorophores for detection. The signal is amplified, as multiple biotin molecules are conjugated to one antibody, subsequently binding streptavidin, which results in greater amount of enzyme or fluorophores than using a conventional enzyme-linked antibody. In addition, the non-covalent bond between streptavidin and biotin occurs rapidly and is very strong and stable. Generally streptavidin is preferred over avidin, as avidin contains sugar residues that can result in non-specific binding. Biotinylated primary antibodies or biotinylated secondary antibodies are frequently used in enzyme-linked immunosorbent assay (ELISA) and in immunoblotting or Western Blot (WB), as well as other applications.

Fluorescein isothiocyanate (FITC)

Fluorophores or fluorochromes are generally comprised of several aromatic groups or cyclic structures and emit fluorescent light when excited by a light source. Fluorescein isothiocyanate (FITC) is one of several fluorophores used in the labeling of antibodies; it is a small organic compound and a fluorescein derivative. FITC is a bright green fluorophore with maximum excitation wavelength of 488 nm and maximum emission wavelength of 530 nm. Fluorescent dyes have characteristic emission spectrums, and those that differ noticeably in their emission spectrums can be combined in multicolor labeling or multicolor imaging, making it possible to distinguish various targets simultaneously in the same assay. This has been extremely useful for protein co-localization studies by cell imaging, and for identifying and isolating specific cell populations in immunology research. FITC labeled antibodies are commonly used in immunofluorescent assays (IF), as well as in flow cytometry (FC), and bead-based multiplex assays, among other applications. Other frequently used fluorochromes include phycoerythrin (PE), cyanine (Cy) and rhodamine, among others.

Horseradish peroxidase (HRP)

HRP is a relatively small forty-four kilodalton glycoprotein and one of the most common enzymes conjugated to antibodies. HRP in the presence of hydrogen peroxide catalyzes the conversion of chromogenic substrates, such as diaminobenzidine (DMB) and tetramethylbenzidine (TMB), into brown or blue colored products, respectively. It also acts on chemiluminescent substrates, such as luminol, emitting light in the process, which can be captured by an X-ray film or camera. HRP-linked antibodies are used in WB, ELISA, and immunohistochemistry (IHC), for detection in colorimetric and chemiluminescent assays. Another popular enzyme often conjugated to antibodies is alkaline phosphatase (AP).

Often researchers experience difficulty in finding primary antibodies already conjugated with these labels, and may opt to conjugate their own antibodies. However, antibody conjugation can be a time-consuming process involving extensive optimization. In addition preservatives, such as sodium azide and BSA, added to the antibody buffers by suppliers may hinder the conjugation process. Non-optimal conjugation may also lead to alterations that may cause reduced antibody binding to the antigen. To address these challenges, EpiGentek offers most of their primary antibodies already conjugated with HRP, FITC or biotin, as well as in unconjugated form, and they are thoroughly validated for use in a number of applications. In addition, EpiGentek also offers several secondary antibodies conjugated with biotin or HRP, for use in ELISA, immunofluorescence, immunohistochemistry and western blot.

Choosing the right antibody label for your immunoassay

Label	Advantages	Immunoassays
Biotin	Amplified signal-great for detection of low abundance targets	ELISA IHC WB
FITC	Multiplex capability-detection of multiple targets simultaneously by combining with other fluorophores	Flow cytometry Immunofluorescence IHC WB
HRP	Low cost-no additional reagents or costly instruments required	WB ELISA IHC

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