Probes labeling with enzymes

As the result of high specificity and sensitivity, nucleic acid probes are in direct competition with immunoassay for the analytes of some types of clinical analytes, such as infectious disease testing. Assays are being developed, however, that combine both probe and immunoassay technology. In such hybrid probe—immunoassays, the immunoassay portion detects and amplifies the specific binding of the probe to a nucleic acid. Either the probe per se or probe labeled with a specific compound is detected by the antibody, which in turn is labeled with an enzyme or fluorophore that serves as the basis for detection. 

Similar techniques can be used to devise avidin—biotin assay systems for detection of nucleic acid hybridization. DNA probes labeled with biotin can be detected after they bind their complementary DNA target through the use of avidin-labeled complexes (Bugawanefrz/., 1990 Lloyd etal., 1990). Direct detection of hybridized probes can be accomplished, in a manner similar to that for LAB, by incubating with an avidin-enzyme conjugate followed by substrate development. BRAB-like and ABC-like assays also can be utilized to further enhance a DNA probe signal (Chapter 17, Section 2.3). 

In the laboratory. DNA can be cut into small pieces by enzymes called restriction endonucleases. The.se enzymes are restricted to cutting the DNA at specific base pair sequences. Tbe presence of polymorphic sites, where there are differences in the base pair sequence of the DNA, may create or abolish a cutting site for a particular restriction endonuclease. In these instances, the polymorphisms are known as restriction fragment length polymorphisms (Rf-LPs) as they lead to the production of DNA fragments of different lengths after enzyme action. DNA fragments may be separated by electrophoresis, transfered to a nylon membrane and hybridized with a DNA probe labelled with a radioactive or optically active marker. This is called. Southern blot analysis (Fig. 1). 

ELISA assays exist in a number of formats. In some cases the reaction product absorbs light, and in other cases the product is strongly fluorescent. The second antdxidy is not always labeled with enzyme but rather is sometimes detected with yet another antibody that contains the bound enzyme. This procedure eliinioates the need to attach probe or enzyme to a specific antibody which may be in short supply. 

Chapters 17-22 describe the hybridization of the nonradioactive probes to the DNA and RNA immobilized on blots, together with the detection systems necessary to reveal where the probe has hybridized. Chapters 17-19 deal with digoxigenin probes, with Chapters 17 and 19 describing chemiluminescent detection on DNA and RNA blots respectively, and Chapter 18 describing a colorimetric detection system. Chapter 20 deals with enhanced chemiluminescent detection of enzymically labeled probes, whereas Chapters 21 and 22 describe enhanced chemiluminescent detection of large (Chapter 21) and small (oligonucleotide. Chapter 22) probes labeled with fluorescein. 

In the first approach, the antibody Abj captures the target microbe, which is interrogated using the antibody labeled with enzyme. The substrate becomes converted to an electro-chemically active product measured at the electrode. In the second approach, the captured microbe impedes the redox probe to access the electrode surface. Impedance (or capacitance, or decrease in the electron transfer) can be directly measured. Label-free approaches are simpler, involve a lower number of steps, and decrease commonly both, analysis time and cost. However, sensitivity usually becomes enhanced when a label (especially when amplification occurs) is involved. In next subsections, both approaches are commented in more detail. 

As an alternative, extremely sensitive detection can be achieved with reporter antibody probes tagged with intensely SERS-active compounds or with enzymes that react with substrates to yield SERS-active products. These methods often involve sandwich immunoassay techniques, which increase the number of required steps but offer the advantages of excellent sensitivity and the potential for label multiplexing. For example, Nie and coworkers recently reported the simultaneous detection of two types of antigens in a... 

Figure 27.1 Three common nucleoside triphosphate derivatives that can be incorporated into oligonucleotides by enzymatic means. The first two are biotin derivatives of pyrimidine and purine bases, respectively, that can be added to an existing DNA strand using either polymerase or terminal transferase enzymes. Modification of DNA with these nucleosides results in a probe detectable with labeled avidin or streptavidin conjugates. The third nucleoside triphosphate derivative contains an amine group that can be added to DNA using terminal transferase. The modified oligonucleotide then can be labeled with amine-reactive bioconjugation reagents to create a detectable probe.

Imaging can also be useful for multiprobe detection, for example using fluorescent probes together with CL reactions. Potentially it is possible to detect first a fluorescent probe, and second detect the CL probe by adding the CL substrate. Probes marked with different CL labels, usually enzymes requiring different substrates, can also be used at the same time, provided the first CL substrate is removed before a second CL analysis is performed by adding another substrate. 

This labeling has been used with success in yeast where 6 of the 17 known DUBs were labeled with UbVS [115]. Incomplete labeling likely results from DUBs that do not act on mono-ubiquitin or where the UbVS could not access the active site. The labeling has also been used with great success in mammalian cell lysates to identify novel ubiquitin DUBs [41]. A novel deneddylating enzyme and a novel DUB that acts on autophagy-related UbL proteins have also been identified using vinyl sulfone labeled probes [26, 37, 116]. 

Common haptens used for labeling DNA probes for BISH assays are biotin, DIG, DNP, FITC, and Texas Red. Based on the size of your DNA targets, you may choose from a direct detection or an indirect detection for BISH assays. In general, an indirect detection system can provide better sensitivity compared to a direct detection system. For an indirect detection, you need to select a combination of two antibodies raised with two different animal species, such as a mouse anti-DIG antibody and a rabbit anti-DNP antibody, so that enzyme-labeled anti-mouse antibody and anti-rabbit antibody can be applied for signal detection. If a direct BISH detection is going to be applied, anti-hapten antibodies raised in the same animal species that are labeled with either AP or HRP enzyme molecules... 

Dioxetanes, labeled with triggers sensitive to the alkaline-phosphatase enzyme, serve as highly sensitive chemiluminescent probes in numerous bioassays. Current applications include immunoassays, membrane-based detection of proteins and nucleic acids, and microplate-based and array-based nucleic-acid detection. ... 

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