Detection system enzyme bridge

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

After establishing a molecular bridge between antigen and the detection enzyme by means of specific antigen-antibody reactions, the antigen-antibody-enzyme complexes are visualized typically by chromogenic reactions. A variety of enzyme-substrate systems have been defined which yield soluble or insoluble, pigmented end products of different colors based on the needs of the researcher. 

The avidin/biotin system is rapidly becoming an important tool for EIA since (i) avidin has an exceptionally high affinity for (+ )-biotin (lO M" ) (ii) (-l-)-biotin is very easily coupled to antibodies and to many enzymes often without any loss of activity and, (iii) avidin is very stable and has several binding sites so that it may be used as a bridging molecule between two biotinylated molecules. The use of the avidin/biotin system produces superior detectabilities and low background levels. 

Scrambling of label from the p non-bridge to the P-y bridge position in ATP would result. The PIX methodology for detection of terminal phosphate transfer was pioneered by Middlefort and Rose (1976), and its application to enzyme systems has been reviewed (Rose, 1979). 

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