Detection of biotin

Biotin (vitamin H, vitamin B7) is not only an important coenzyme, but also a laboratory tool to label particular antibodies or other proteins of interest with intention to detect them by secondary/tertiary reagents or to immunoprecipitate them. These detection systems can be based on streptavidin/avidin or on an antibody. Streptavidin is a homotetrameric protein with high affinity to biotin. Strong interaction between these molecules can be used for preparing of streptavidin-biotin complexes, which represent a signal amplifying system resistant to commonly used detergents and changes in temperature or pH. Fluorescently labeled or HRP labeled streptavidin can be used as a secondary reagent also alone. Another system is based on anti-biotin antibody. It can be used either directly in conjugated form, or unconjugated as a signal amplifier, followed by conjugated secondary or tertiary reagent. Compared to streptavidin-biotin interaction, the interaction between anti-biotin antibody and biotin is weaker, which can be used for easier release when elution is needed. Concerning immunohistochemistry, high content of natural biotin in some tissues, e.g. kidney or liver, can cause undesired background signal of biotin detection systems, especially in case of frozen sections. Preparation of formalin-fixed paraffin-embedded sections significantly reduces this background signal.
 
 

Fig. 1: As demonstrated here, detection of biotin can have more arrangements.
 
 
Mouse monoclonal antibody 1D4-C5 recognizes biotin and can be used to detect biotinylated antigens or biotinylated antibodies in various applications.
 
Available formats:
purified                11-944-C100
HRP                       coming soon
PE                          coming soon
 
 
Streptavidin conjugates:
 
Available formats:
FITC                       EXB0041
PE                          EXB0042
APC                       EXB0043
 
 

Fig. 2: ELISA analysis of a biotinylated antigen using biotin-specific antibody and streptavidin. Microtitration wells were coated with biotinylated human IgG (0.01 μg/ml) and blocked. Then either anti-biotin mouse monoclonal 1D4-C5 (A, C) or streptavidin-HRP (B) was added in six different doses. Finally, goat anti-mouse-HRP conjugate (A) or streptavidin-HRP (C) was added. HRP signal increased in dose dependent manner, but with different dynamics, when biotinylated antibody was detected by anti-biotin mAb/goat anti mouse-HRP (A) or by streptavidin-HRP (B), and it was falling to zero in dose dependent manner when streptavidin-HRP was competed by anti-biotin mAb 1D4-C5 (C).
 
 

 
Fig. 3: Flow cytometry surface staining pattern of human peripheral whole blood stained using anti-human CD4 (MEM-241) Biotin antibody and secondary stained using Streptavidin PE (concentration in sample 1 μg/ml).
 
 
 
 
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