Stem Cells Labeled With Fluorescence dye molecules

About Secondary Antibodies

Secondary antibodies bind to the unconjugated primary antibody to detect the antigen that the primary antibody is bound to. Secondary antibodies are usually purified using an antigen affinity purification system where the antigen attached to the chromatography matrix is an immunoglobulin of the same Ig class as the primary antibody. Sometimes these secondary antibodies which are actually polyclonal in nature, must be absorbed by additional chromatography techniques to remove potential cross reactivity to other antibodies or serum proteins from other animal species. There is sometimes an advantage to using the indirect secondary antibody system to detect primary antibodies because a signal amplification occurs because multiple secondary antibodies can bind to a single primary antibody.

Secondary Antibody binding to primary antibody - direct vs. indirect method

Advantages of Secondary Antibodies 

  • Sensitivity – Increased sensitivity due to signal amplification from multiple secondary antibodies binding to the single primary antibody. 
  • Versatile – A secondary antibody can be used to detect many different unconjugated primary antibodies without having to conjugate each primary antibody to a separate reporter molecule. 
  • Flexibility – Secondary antibodies with the specific specificity for the primary antibodies of common species are widely available pre-conjugated to many common reporter molecule (fluorescent and enzyme conjugates) options. 
  • Dynamic – In some cases the same secondary antibodies can be used across applications to validate target antigen detection such as fluorescent western blot and immunofluorescence. 
  • Multiplexing – Secondary antibodies may offer the ability to perform multi-labeling  experiments for immunocytochemistry and immunohistochemistry and is therefore extremely useful in examining the behavior and interactions between cellular proteins.  

About Streptavidin

Streptavidin is a tetrameric bacterial protein isolated from the bacterium Streptomyces avidinii. Like avidin from egg whites, streptavidin can bind four molecules of biotin and has one of the highest noncovalent affinity constants known in nature (Kd ~ 10-15). Streptavidin is composed of four identical subunits with a molecular weight of approximately 53,000 daltons. Streptavidin is free of carbohydrate side chains and has a near neutral isoelectric point of 6.5. 

Streptavidin has a wide variety of applications such as use as a second step reagent with biotin labeled primary antibodies or in the formation of class I and II MHC tetramers for detection of antigen specific T-cells. Leinco Technologies offers streptavidin conjugated to a wide spectrum of fluorescent dyes and enzymes for use as a second step reagent for detection of biotin labeled primary antibodies and other biotin labeled molecules. The advantage of using streptavidin is the amplification of signal achieved due to binding of multiple streptavidin conjugated complexes to one biotin labeled primary antibody. Or in the case of a tetramer, the binding of multiple biotinylated immune system molecules to one streptavidin to increase detection sensitivity of antigen specific T-cells.

The Avidin-Biotin Complex Process
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