Biotin-mediated signal amplification in IHC

Biotin (= Vitamin H) is a stable, relatively small molecule that non-covalently binds to avidin (a protein from egg white) or streptavidin (a protein isolated from Streptomyces avidinii). This interaction occurs with high affinity and can be used for signal amplification in immunoassays.

Enzym conjugated secondary antibodies - Biotin

Biotin-SP (long spacer) is the trade name of Jackson ImmunoResearch for Biotin with a long spacer (6 atoms) that is located between biotin and conjugated protein (see image). The combination of Biotin-SP conjugated secondary antibodies and streptavidin-alkaline phosphates (ALP) or streptavidin-horseradish peroxidase (HRP) can increase sensitivity in enzyme-immunosassays (EIA) compared to directly conjugated (without spacer) antibodies. The linker (22.4 Ä) extends the space between secondary antibody and biotin, making the protein surface more accessible for streptavidin-enzymes.

Biotin-SP molecule. Molecular weight 454,54 Da, Linker 22,4 Ä. (Image: Jackson ImmunoResearch)
IHC staining using biotin-conjugated Donkey IgG anti-Rabbit IgG (H+L) (711-065-152) secondary antibody, followed by HRP-conjugated Streptavidin (016-030-084). Signal was visualized using DAB substrate. (Image: Jackson ImmunoResearch)

Additional reagents are required for the visualization of biotin-conjugated secondary antibodies. We offer streptavidin and mouse anti-biotin antibodies conjugated to fluorescent dyes or enzymes.


  • Jackson ImmunoResearch: Catalogue 2019
  • Mulisch, M. & Welsch, U. Romeis – Mikroskopische Technik. (Springer-Verlag, 2015).