TechTip: Biotin and IHC Detection
Biotin and IHC Detection
Biotin (or vitamin H) is a water-soluble B-complex vitamin that is required by all organisms. Biotin is present in a variety of tissues, with high concentration in the intestine, brain, kidney, spleen, liver, lung, and adipose tissue.
Biotin's interaction with avidin and streptavidin is of particular importance in immunohistochemistry (IHC). Avidin is a 68 kDa tetrameric glycoprotein composed of four identical subunits. Each subunit is glycosylated, and contains one binding site for biotin. Streptavidin is a neutral bacterial analog of avidin which occurs in a non-glycosylated form. Due to its glycosylation state, non-specific binding of biotin by avidin has been observed. Streptavidin and avidin both possess great affinity for biotin, with which they form irreversible bonds. This binding can be exploited in a number of biochemical assays, particularly immunohistochemistry.
The LAB-SA (labeled [strept]Avidin-Biotin) method for IHC detection uses an affinity-purified, biotinylated secondary antibody and a streptavidin-enzyme conjugate to detect a primary antibody bound to a cell or tissue antigen. A chromogenic reaction is then performed to visualize the location of the antigen/antibody/enzyme complex. Invitrogen Histostain®-SP, Histostain®-SAP, Histostain®-Plus, Histostain®-DS, HistoST5050™, and Cap-Plus™ detection kits all utilize LAB-SA technology.
One potential problem in an IHC assay may arise when labeled streptavidin binds to endogenous biotin. This binding produces excessive background staining and false-positive reactivity. This situation may be rectified by using Endogenous Avidin/Biotin Blocking Kit (Cat. No. 00-4303), or SuperPicTure™ polymer detection kit (Cat. No. 87-9663 or 87-9963). Paraffin-embedding of tissues destroys or masks much of the endogenous biotin activity, but Heat Induced Epitope Retrieval (HIER) can retrieve and unmask endogenous biotin.1 As temperature, time, and pH increase during tissue pretreatment, access to and reactivity with endogenous biotin increases. Frozen tissues will always exhibit high levels of endogenous biotin. Endogenous Avidin/Biotin Blocking Kit is designed for use with LAB-SA detection kits. SuperPicTure™ polymer kit avoids endogenous biotin reactivity, employing a polymer conjugate that contains multiple enzymes and Fab fragments.
Shi ZR, et al. J Histotechnology 23(4):327-331, 2000.