Signal Amplification Using Chromogenic Detection

Chromogenic detection uses enzymes to convert soluble substrates to insoluble, chromogenic end products at the site of antigen expression. The two most common enzymes used for chromogenic detection are Horseradish peroxidase (HRP) and alkaline phosphatase (AP); HRP converts 3,3’ diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), into brown and red end products, OriGene offers two types of detection systems: i) Traditional detection systems and ii) Polymer-linked detection systems.

Browse all traditional IHC Kits Browse all Polymer-Linked IHC Kits

Streptavidin-Biotin Complex (ABC) Method

The streptavidin-biotin complex (ABC) detection method is one of the most widely used methods for staining. Streptavidin is a tetramer with a high affinity for biotin; each streptavidin molecule can bind up to four biotin molecules. In the ABC system, streptavidin and biotinylated enzymes are combined to form a large complex that binds to a biotinylated target, usually a secondary antibody. The multilayer complex formed amplifies the signal generated by the antigen-antibody interaction, making it easier to detect and visualize low-abundance antigens. Usually, a secondary antibody conjugated with the complex attaches to tissue-bound primary antibodies; a substrate is then added and converted into a colored product to mark the target antigen.

Polymer Linked Detection Method

Polymer-based immunohistochemistry (IHC) methods are alternative techniques to the traditional streptavidin-biotin complex method. These polymer-based methods utilize enzyme-labeled polymer conjugates to amplify the signal generated by antigen-antibody interactions, similar to the streptavidin-biotin complex method. These methods offer increased sensitivity, reduced background staining, and simpler protocols.

Advantages Vs Disadvantages of Different Detection Method

Advantages Drawbacks
ABC Method
  • Cost-effective
  • Better sensitivity compared to indirect method
  • Ideal for detection of antigen which are moderately or highly expressed.
  • Smaller complex with greater tissue penetration
  • Endogenous biotin in the tissue can produce higher background. Pre-blocking step is recommended. But, tissue with higher presence of biotin like liver and kidney can still produce higher activity.
  • Poor reproducibility
  • Not efficient to detect antigen with lower expression levels
  • Reduced sensitivity
  • Complicated protocols
Polymer Linked Method
  • Higher Sensitivity and Specificity
  • Lower Background
  • Reduced steps compared to ABC and B-SA method
  • Higher cost
  • Less versatile

Selection Guide for OriGene IHC Detection Kits

Detection System Enzyme Cost/ Assay Biotin-Free Polymer Sensitivity Amplification Ready to use format Typical number of steps
ABC System HRP or AP + No - ++ ++ Blocking step required 7
Polink 1 HRP or AP ++ Yes + ++ ++ Yes 5
Polink 2 HRP or AP ++ Yes + +++ +++ Yes 6
Polink 2 Plus HRP or AP +++ Yes + ++++ ++++ Yes 6
MultiStain Kits HRP or AP ++ Yes + +++ +++ Yes 7-15
Klear Species Kit HRP or AP ++ Yes + ++++ ++++ Yes 7
Assay Kits Resources