Conjugated Primary Antibodies

A conjugated antibody is a polyclonal or monoclonal antibody that has a molecule attached which can be used to create a detectable signal. Detection can be visualized by color-generation, fluorescence, or other signals. Conjugated antibodies are used in a wide range of applications including flow cytometry, immunohistochemistry, immunocytochemistry, western blot, and ELISA.

Types of Detection Methods

There are two types of detection methods used with antibodies, direct and indirect.

Direct detection uses a primary antibody that is directly conjugated to a label. By conjugating primary antibodies, you can directly detect the target without use of secondary antibodies. The advantages of direct detection include ease of use for multicolor staining and eliminating the concerns regarding non-specific binding of the secondary antibody. It is ideal to use directly conjugated antibodies when the target of interest is in high abundance, or the conjugate signal can compensate for low target expression levels.

Indirect detection uses a primary antibody that is unconjugated and a conjugated secondary antibody that is specific for the host species of the primary antibody. Indirect detection is the preferred method if the target of interest has a lower level of expression. By using secondary antibodies, this allows signal amplification due to the secondary antibodies carrying multiple labels that can bind to the primary antibody. In general, indirect detection methods generally have a higher level of sensitivity and generate a more intense signal.

Antibodies can also be conjugated to a label that provides chromogenic detection instead of fluorescence. When an antibody is conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase, the enzyme reacts to its substrate and an insoluble colored product is localized to the sites of antigen expression. Chromogenic labeled antibodies are great options for ELISA , Western blot, and Immunohistochemistry.

Biotin conjugated antibodies are recommended to be used when target expression is low. When a biotinylated antibody is used, the signal can be significantly amplified by subsequent incubation with a labeled streptavidin-biotin (LSAB Method). This due to the ability of streptavidin to bind up to four biotins per molecule. Fluorescent or chromogenic conjugated streptavidin is used to detect the biotin on the primary antibody and produce a signal.

NOV/CCN3 was detected in immersion fixed paraffin-embedded sections of human prostate using Goat Anti-Human NOV/CCN3 Biotinylated Antigen Affinity-purified Polyclonal Antibody (Catalog # BAF1640) followed by Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008).

ICAM-1/CD54 was detected in rat liver tissue using Goat Anti-Rat ICAM‑1/CD54 Biotinylated Antigen Affinity-purified Polyclonal Antibody (Catalog # BAF583) followed by Streptavidin-HRP (Catalog # DY998).

An alternative option to the biotin-streptavidin amplification complex, is R&D Systems’ VisUCyte^™ HRP Polymer. The VisUCyte^™ HRP Polymer is a biotin-free detection reagent that eliminates the need to perform additional quenching of endogenous biotin and avidin present in some tissues. With fewer blocking and incubation steps, researchers can achieve specific staining in cells and tissues faster compared to the traditional streptavidin-biotin-HRP procedure.

VisUCyte^™ HRP Polymer

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