Conjugated Secondary Antibody: A Guide

Conjugated Secondary Antibody: A Guide

by: ImmunoReagents University (IRU)

Conjugated Secondary Antibody: A Guide is an article designed to help you learn and select the appropriate conjugated secondary antibody. Secondary antibodies are conjugated (AKA-Labeled) to a large number of labels, including enzymes, biotin, and fluorescent dyes/proteins.  At times the choices seem overwhelming, but this guide should make that decision easier. ImmunoReagents manufacturers a wide variety of conjugated antibodies that will help with your immunoassay.


Choosing the best label depends upon the immunoassay that needs to conducted.


Table 1- Choose the ImmunoAssay






Enzymes – are proteins that act as catalysts to drive a chemical reactions. Enzymes that are used in immunoassays react with other chemical called substrates.  When the enzyme labeled antibody comes in contact with the substrate a enzymatic reaction occurs. The reaction produces a color change that can be visualized.

The most widely used enzymes are:

  • Horseradish Peroxidase (HRP) – HRP is a glycoprotein enzyme derived from the horseradish plant. HRP reacts with a variety of substrates. Typically the HRP enzyme oxidizes peroxide and elicits a color change. The type of substrate will be determined by the detection method. For example, if the color change will be a chromogenic substrate will be used such as TMB, DAB, or ABTS. If the HRP will be used to emit a light in chemiluminescent assays a luminol based substrate will be used.
  • Alkaline Phosphatase (AP) – is an enzyme used to remove phosphates by means of dephosphorylation.  It can be visualized by using a colorimetric substrate pNNP.

IR Tip-Due to the large size of Alk-Phos conjugates they are typically not used in immunohistochemistry because they are difficult to penetrate the cells. 

Biotin/Streptavidin – is commonly used to detect proteins of interest that are in very low levels and cannot be detected using traditional secondary labeled antibodies. Biotin/Streptavidin detection system can offer a 6-10 fold signal enhancement. Biotin binds to Streptavidin and is one of the strongest natural bonds. Neither Biotin or Streptavidin elicit a color change.  This is achieved by adding an enzyme/dye to the Streptavidin molecule. In general, secondary antibodies are labeled with biotin. Since biotin is a small molecule many biotin molecule can fit on a single antibody. By having more biotin molecules per antibody, this allows more labeled Steptavidin molecules to bind. Consequently, the signal is amplified.

IR Tip- It is common for the biotin labeled secondary antibody to be added first, followed by the labeled Streptavidin conjugate.

Fluorescent Conjugated Secondaries – secondary antibodies conjugated to fluorescent labels/dyes are widely used for multi-labeling experiments. Due to the unique excitation/emission profiles, fluorochromes have novel characteristic spectra for absorption and emission.  Fluorescent dyes are excited at a particular wavelength and emit a photon at another wavelength.  Tandem dyes are two dyes. One dye is a donor and the other dyes is an acceptor fluorochrome molecule. The dyes are in close proximity, which allows for energy transfer between the two.  The tandem dye is excited at the excitation wavelength of the acceptor molecule and emits a photon at the emission wavelength of the donor molecule.

ImmunoReagents currently offers a vast range of DyLight® conjugates which are ideal for multi-color analysis that span the whole of the spectrum from the UV to far infra-red.  Furthermore, the DyLight® dyes are conjugated to secondary antibodies that have been cross-absorbed against various species. Read more about ImmunoReagents’ DyLight Antibodies: DyLight Flyer

Fluorescent Poster


ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulins, primary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina.