Immunohistology and immunocytology techniques permit the specific demonstration of cell and tissue antigens in tissue sections, cell smears and cytospins using enzyme or fluorescence as a label.
The IMMU-MARK immunostaining kits are based on the biotin-streptavidin amplified methodology. Streptavidin is a 60,000 dalton avidin analog isolated from the bacterium Streptomyces avidinii1. Like avidin isolated from egg whites, (molecular weight approximately 68,000) streptavidin is capable of binding the small, water soluble vitamin biotin (molecular weight 244) with an unusually high affinity (KD = 10-15). Although the binding between streptavidin and biotin is non-covalent, it is over one million times stronger than antigen-antibody interactions and is essentially irreversible.
Unlike avidin, streptavidin exhibits very little non-specific binding to normal tissue, e. g., mast cells, kidney, liver and brain tissue at physiological pH1,2. A high degree of non-specific staining with avidin occurs because (1) of the high isoelectric point of avidin which increases electrostatic interaction and (2) the high carbohydrate content of avidin which accounts for non-specific binding to tissue proteins. Streptavidin binds to biotin as effectively as avidin but its isoelectric point is close to neutral and contains no carbohydrate residues. Therefore, the problem of non-specific binding is eliminated.
The streptavidin-biotin system represents the latest technology in an effective and sensitive immunoassay for demonstration of antigens in human and animal tissue sections, cell smears and cytospins. The system gives clear, intense staining with a minimum of background. This eliminates the need for blocking to suppress endogenous biotin activity. In this amplification system the enzyme is directly conjugated to the streptavidin, thus providing highly stable reagents.