Pierce Streptavidin Poly-HRP Conjugate

Thermo Scientific Pierce Streptavidin Poly-HRP Conjugate is biotin-binding protein conjugated with polymers of horseradish peroxidase, enabling signal amplification and detection of biotinylated antibodies for IHC and other methods.

This poly-HRP conjugate is designed to deliver the highest sensitivity and low background in immunoassays where sample volume is limited or when the target molecule is present at low levels. Pierce Streptavidin Poly-HRP is purified to remove unconjugated streptavidin molecules that reduce signal intensity by competing for binding sites with horseradish peroxidase (HRP)-conjugates molecules. In addition, the conjugate is devoid of HRP monomers that can cause background signal. The Pierce Streptavidin Poly-HRP is compatible with chromogenic, fluorogenic and chemiluminescent HRP substrates used in ELISA, Western blotting, immunohistochemistry (IHC) and nucleic acid hybridization assays.

Highlights:

• High sensitivity – detect low-abundant targets (low picogram-femtogram range) with high signal-to-noise ratios
• Robust – consistent manufacturing and purification to eliminate low- and un-conjugated molecules for minimal background and high sensitivity
• Flexible – compatible with chromogenic, fluorogenic and chemiluminescent substrates
• Versatile – compatible with ELISA, Western blotting and immunohistochemistry (IHC)
• Easy to use – can be directly substituted into immunoassays and other detection assays
• Convenient – ready-to-use stabilized liquid format stored at 4°C
• Cost effective – requires less conjugate per assay and standard HRP conjugates

Product Details:

Thermo Scientific Pierce Streptavidin Poly-HRP Conjugate enables highly sensitive ELISA detection. Pierce Streptavidin Poly-HRP, Supplier S poly-HRP, and a commercially available nonpolymerized streptavidin-HRP conjugate (non poly-HRP) were compared in an IFNγ ELISA. A 96-well polystyrene microplate was coated with an anti-Human IFNγ antibody, blocked and INFγ standards prepared using the reagents and protocol from the Thermo Scientific Human IFN gamma Screening Set (Part No. ESS0002). The biotinylated anti-human IFNγ detection antibody was probed with the 3 different streptavidin-HRP conjugates (50ng/mL) and the assay was developed according to the protocol.

Several forms of streptavidin-HRP conjugates are available that do not contain polymerized HRP (poly-HRP) and are suitable for most biotin detection assays. These and other commonly used HRP-conjugated antibodies and biotin-binding proteins are limited to one to three HRP molecules per protein in order to maintain functionality of the conjugate. This molar ratio of conjugation is effective in routine experiments but may lack the sensitivity required for detecting targets present in low picogram to femtogram amounts without the use of additional steps to amplify the signal.

When a higher degree of signal amplification is needed with standard HRP conjugates, methods such as peroxidase anti-peroxidase (PAP) systems or avidin-biotin complexes (ABC) can be used. However, these methods increase the cost of the assay and the number of steps that must be optimized. Pierce Streptavidin Poly-HRP circumvents such problems because it uses polymers of HRP molecules that are conjugated to streptavidin in a proprietary manufacturing process that increases the molar ratio of HRP on the conjugate while maintaining functionality. The increased molar ratio of HRP molecules to streptavidin results in more HRP molecules being available per binding event to react with the substrate. Therefore, Pierce Streptavidin Poly-HRP provides substantial signal amplification over other HRP-conjugates, eliminating the need for additional steps and saving time and expense.

Thermo Scientific Pierce Streptavidin Poly-HRP produces IHC images with outstanding clarity. Mounted tissues were incubated overnight at 4°C with biotinylated anti-mouse cytokeratin 18 at 18.6µg/mL. The negative control tissue was incubated with blocking buffer only. Tissues were incubated with 4µg/mL of either Pierce Streptavidin Poly-HRP or a similar product from Supplier S.

As with all immunoassays, optimization is required to achieve the best signal-to-noise ratio. Switching to higher sensitivity substrates and conjugates can reveal more desired signal as well as background signal. If this occurs, follow standard primary and secondary dilution techniques or perform blocking buffer optimization.

Related Resources:

Protein Methods Library: Overview of Immunohistochemistry (IHC)
Protein Methods Library: Immunodetection Strategies for IHC

Related Products:

All Biotin-Binding Proteins and Conjugates
Anti-Mouse and Anti-Rabbit IgG Poly-HRP Conjugates
ABC Staining Kits