Detection system biotin-streptavidin

The presence of biotin labels on an antibody molecule provides multiple sites for the binding of avidin or streptavidin. If the biotin binding protein is in turn labeled with an enzyme, fluoro-phore, etc., then a very sensitive detection system is created. The potential for more than one labeled (strept)avidin to become attached to each antibody through multiple biotinylation sites provides an increase in detectability over antibodies directly labeled with a detectable tag. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

All detection systems can be employed following HER and noticeable improvement of most antigen detection will be observed. However, we recommend using sensitive detection systems, such as streptavidin-biotin-peroxidase methods when antigen density is low. 

For greatest sensitivity, the biotin-streptavidin system should be used in antibody detection, especially since proteins separated in 2D are often more difficult to detect than when separated in ID... 

NG Hentz, LG Bachas. Class-selective detection system for liquid chromatography based on the streptavidin-biotin interaction. Anal Chem 67 1014-1018, 1995. 

In glycoprotein detection systems the carbohydrate portions of proteins are oxidized with sodium metaperiodate to generate aldehydes that can react with hydrazides. A biotin hydrazide is used to attach biotin onto the oxidized carbohydrates and horseradish peroxidase-conjugated streptavidin is used for chemiluminescence-based detection (Glycoprotein Detection Module, Amersham Biosciences, Uppsala, Sweden). 

A protein-binding assay (BA) coupled with hplc provided a highly sensitive post-column reaction detection system for the biologically important molecule biotin and its derivative biocytin, biotin ethylenediamine, 6-(biotinoylamino) caproic acid, and 6-(biotinoylamino)caproic acid hydrazide (71). This detection system is selective for the biotin moiety and responds only to the class of compounds that contain biotin in their molecules. In this assay a conjugate of streptavidin with fluorescamine isothiocyanate (streptavidin—FITC) was employed. Upon binding of the analyte (biotin or biotin derivative) to streptavidin—FITC, an enhancement in fluorescence intensity results. This enhancement in fluorescence intensity can be directly related to the concentration of the analyte and thus serves as the analytical signal. The hplc/BA system is more sensitive and selective than either the BA or hplc alone. With the described system, the detection limits for biotin and biocytin were found to be 97 and 149 pg, respectively. 

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

Biotin has served this purpose well in both nucleic acid and antibody probe systems. As well as being easily detected with immunoglobulins specific for biotin, biotin may also be detected non-immunologically with avidin or streptavidin, two proteins which share a marked, highly specific affinity for biotin. The affinity constant for avidin-biotin interactions is approximately 10 - liters/mole, much higher than the range for antigen-antibody interactions which are commonly between 10 -10 liters/mole. Consequently, a vast number of detection complexes composed of avidin or streptavidin bound to a detection system are commercially available (e.g. streptavidin-alkaline phosphatase). 

While enzymes may be covalently attached directly to primary probe molecules, as noted above for reasons of reagent versatility, steric factors, and potential signal amplification, indirect detection systems appear to be the more popular. Consequently, enzyme-probe conjugates are typically complexes of a desired enzyme marker and a secondary level probe that is, a probe molecule that can specifically identify a primary level probe molecule, such as an alkaline phosphatase-streptavidin conjugate can identify a biotinylated nucleic acid probe by virtue of the binding affinity between streptavidin and biotin. Other examples of enzyme-probe systems are given in the preceding section on direct and indirect detection systems. 

A DNA optical sensor system was proposed by Cass and co-workers [35] based on the combination of sandwich solution hybridization, magnetic bead capture, flow injection and chemiluminescence for the rapid detection of DNA hybridization. Sandwich solution hybridization uses two sets of DNA probes, one labelled with biotin, the other with an enzyme marker and hybridization is performed in solution where the mobility is greater and the hybridization process is faster, rather than on a surface. The hybrids were bound to the streptavidin-coated magnetic beads through biotin-streptavidin binding reaction. A chemiluminescence fibre-optic biosensor for the detection of hybridization of horseradish peroxidase-labelled complementary DNA to covalent immobilized DNA probes was developed by Zhou and co-workers [36]. 

For quantitation of HIV-1 virus load in serum/plasma samples, testing samples in duplicate is recommended. Samples are considered positive and are quantifiable when duplicate test results are positive. Samples with negative or discordant duplicate results are considered Amp-RT negative. Quantitative detection of Amp-RT PCR amplicons is made by an ELISA-based, nonradio-active oligoprobing system. The system uses streptavidin-coated microtiter wells that capture the biotin-labeled PCR product (DIG-detection ELISA). The protocol for the ELISA is as follows ... 

Despite the wide use of radioprobes in colony or plaque hybridization assays, nonradioactive probes can be advantageous. The use of biotinylated probes, initially the most common among nonradioactive detection systems, is limited since biotin-streptavidin systems tend to give high background levels with bacterial material unless specific measures are taken. The more recently developed DIG (Table 7.2), but also other hapten-antibody systems such as sulfonated probes, are very attractive alternatives. The main restriction is that monoclonal antibodies (commercially available) should be used since polyclonal antisera often contain antibodies against bacteria. The main drawback of nonradioactive probes is the ability to reprobe the same membrane. It is possible, however, to strip a membrane of its probe after a colorimetric detection and to perform a chemiluminescent detection or vice versa. 

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