Principle of Assay
This product is intended for qualitative immunohistochemistry with normal and neoplastic formalin-fixed, paraffin-embedded tissue sections, to be viewed by light microscopy. This kit is designed to label specific primary antibodies immunohistochemically on tissue sections. These reagents were tested and quality controlled using tissue sections, however, they can also be optimized for cell smears and cytospin preparations. The high affinity of the non covalent interaction between biotin and streptavidin (1x10^15) forms the basis for this immunostaining kit. It requires the formation of an irreversible and specific linkage between biological macromolecules. The immunohistochemical applications of the interaction between avidin and biotin were introduced by Bayer et al. (1979), who described techniques for generating active biotinyl compounds such as biotin-N-hydroxysuccinimide and biotin hydrazine and for conjugating them to various organic compounds, including immunoglobulins and horseradish peroxidase (HRP). Streptavidin (SA) is a tetrameric protein (mol. wt. 4x15,000), isolated from the actinobacterium Streptomyces avidinii (Chaiet & wolf, 1964). Streptavidin can bind to four molecules of biotin. Streptavidin gives superior results as compared to avidin because its isoelectric point is closer to a neutral pH, whereas avidin is positively charged at a physiological pH. Streptavidin does not carry any carbohydrate side chain, whereas avidin is composed of 70% of carbohydrate. Because of these reasons, SA does not have the tendency to bind non-specifically. Primary antibodies bind to target antigens in the tissue sections. The conjugated secondary antibody binds specifically to these receptor antibodies. Biotin conjugated second antibody, in turn, is traced by a streptavidin conjugated enzyme and can be visualized by an appropriate substrate. Immunoperoxidase techniques are spreading rapidly and the practice of anatomic pathology has undergone a revolutionary change since the development of these procedures (Nadji & Morales, 1983). Because of their versatility, sensitivity, and specificity, immunoperoxidase stains are invariably the best stains when and if appropriate antibodies are available. All immunohistochemical techniques require the specific antibody employed to be so labeled that they can be easily seen when attached to cellular antigens. At the same time the sensitivity of immunoperoxidase techniques are central to wide variety of specific antigen localization. Various investigators (Petrusz et al., 1983; Nagle et al. 1983; Giorno et al., 1984) have shown that direct SA-HRP conjugate technique is 4 to 8 times more sensitive than the avidin-biotin complex described by Hsu et al. (1981). Our kit is based on direct SA-HRP conjugate technology. The linker reagent is a cocktail of biotinylated anti-mouse and anti-rabbit, capable of labeling primary antibodies raised in mouse and rabbit.
