Labeled detection reagent

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

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. 

Figure 7.21 Dendrimers that are fluorescently labeled as well as biotinylated create enhanced detection reagents for use in (strept)avidin-biotin-based assays. Large complexes containing multiple fluorescent dendrimers can bind to antigens and form a highly sensitive detection system that exceeds the detection capability of fluorescently labeled antibodies.

Colloidal gold-labeled (strept)avidin can be used as highly sensitive detection reagents for microscopy techniques (Cubie and Norval, 1989) (Chapter 24). Finally, cytotoxic substances coupled to (strept)avidin can be used to direct cell-killing activity toward a tumor-cell-bound, biotinylated monoclonal antibody (or other targeting molecule) for cancer therapy (Hashimoto et al, 1984) (Chapter 21). 

There are mainly three types of transducers used in immunosensors electrochemical, optical, and microgravimetric transducers. The immunosensors may operate either as direct immunosensors or as indirect ones. For direct immunosensors, the transducers directly detect the physical or chemical effects resulting from the immunocomplex formation at the interfaces, with no additional labels used. The direct immunosensors detect the analytes in real time. For indirect immunosensors, one or multiple labeled bio-reagents are commonly used during the detection processes, and the transducers should detect the signals from the labels. These indirect detections used to need several washing and separation steps and are sometimes called immunoassays. Compared with the direct immunosensors, the indirect immunosensors may have higher sensitivity and better ability to defend interference from non-specific adsorption. 

Detection reagent (substrate) for enzyme-labeled antibody 2,2-azino-di(3-ethyl-benzthiazohne sulfonate-6) (ABTS) 0.3 g/L in 0.15 M sodium citrate with 0.1% H2O2 for the detection of horseradish peroxidase-labeled secondary antibody or p-nitrophenyl phosphate (pNPP) 1 g/L in 1M diethanolamine in water for the detection of an alkaline phosphatase-labeled antibody (Kirkegaard and Perry Laboratories). 

ZytoVision ZytoDot2C GISH Implementation Kit contains all necessary BISH detection reagents for the detection of DIG-and DNP-labeled DNA probes on formalin-fixed tissue sections. The probes can be purchased from ZytoVision or other vendors. 

Dako DuoCISH Kit contains HRP- and AP-based immunological detection reagents for Texas Red- and FITC-labeled probes. This is an ideal kit for manual dual-color BISH assay detection. 

Biotinylated enzymes can be used as detection reagents in avidin—biotin assay procedures. Particularly, in the bridged avidin—biotin (BRAB) approach or the ABC technique (Chapter 13, Section 2), a biotin-labeled enzyme is used as the signaling agent... 

This chapter presents an approach to perform enzyme linked immunosorbent assays (ELISA) in a microfluidic format with electrochemical detection. This field of analytical chemistry has shown a strong activity in recent years, and many reports have presented the use of capillary-sized reactors for running immunoassays either in homogeneous format (where the antigen-antibody complex and the labelled revelation reagents are separated prior to detection, as for instance by capillary electrophoresis [1-3]) or in heterogeneous format (where the antibody is immobilised on the inner surface of the microsensor device [4] or on microbeads [5,6]). 

Controls As with any technique it is essential to perform a number of controls. A positive control section should be included with each batch, to test for variations in the intensity of staining from day to day. For each test section, an appropriate immunohistochemical control should be performed to test for the presence of endogenous enzyme activities and/or nonspecific binding of the detection reagents. This is most easily achieved by the exclusion of the enzyme or the labeled nucleotide from the TUNEL/ISEL reaction mixture. 

The drink commonly known as Cherry Water contains water, but also up to 40% volume of ethanol besides the cherry juices. One really cannot call this solution water . Dissolving sugar in water leads to a colorless sugar solution and one should not call it sweet water . In chemistry laboratories, we call some detection reagents limewater and baryta water one should better use more accurate and meaningful labels like calcium hydroxide solution or barium hydroxide solution ... 

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