Sandwich hybridization

DNA probing can be done not only on a membrane, but also on solid supports. An example is the classical sandwich hybridization assay, which uses a capture... 

Hybridization can also be performed in solution phase. Since the capture probe is in solution, the kinetics of hybridization is faster than when the capture probe is immobilized. In the solution phase hybridization format, the capture probe is labeled with an affinity label such as biotin that captures the sample target sequence. The labeled probe then binds to the sample target sequence to form the sandwich. After the hybridization is complete, the sandwich is transferred to an affinity support such as avidin or streptavidin, which will capture the sandwich through the biotin-labeled capture probe. Sandwich hybridization performed in solution followed by capture on an affinity support has been referred to as affinity-based hybrid collection (Kl). 

Amplification of nucleic acids also include RNA amplifications. For instance, the heat-shock mRNA (103-mer) in viable Cryptosporidium parvum in water treatment plants was detected after amplification by nucleic acid sequence-based amplification (NASBA). After amplification, a sandwich hybridization assay was subsequently conducted within a PDMS channel [955]. 

Figure 20. Schematic representation of the analytical protocol of the biosensor (a) Dual hybridization event of the sandwich hybridization assay, leading to the capture of the CdS-loaded CNT tags in the microwell (b) dissolution of the CdS tracer (c) stripping voltammetric detection of cadmium at a mercury-coated glassy carbon electrode. PI, DNA probe 1 T, DNA target P2, DNA probe 2. From reference 114.

Kwoh DY, Davis GR, Whitfield KM, ChappeUe HL> DiMichele LJ, Gingeras TR. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad SciUSA 1989 86 1173-7. 

Fig. 8.3. Sandwich hybridization consists of two steps hybridization in solution with nonoverlapping labeled and haptenated probes (I) and the trapping of the hybrid (via the haptens) on an affinity matrix and washing so that labeled probe is only retained when the target is present (ID.

Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986 Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about 10 molecules with a linear range to at least 10 molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important). 

Sandwich hybridization followed by immunological capture and detection... 

The Probelia PCR System E. coli 0157 H7 (Biocontrol System) use primers and probes specific for E. coli 0157 H7, a synthetic DNA as LAC and a revealing system on microtiter plates. After PCR amplification of the target, the detection is performed by sandwich hybridization. The specificity of this method was assessed on a collection of 98 STEC and 40 non-STEC bacterial strains and detection of 1-10 cfu/25 g... 

Liposome-Enhanced Lateral-Flow Assays for the Sandwich-Hybridization Detection of RNA... 

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

Fig. 5. Sandwich-hybridization assay (A) A biotinylated DNA oligonucleotide, which is complementary to one portion of the target sequence, is mixed with streptavidin and applied to form the capture zone 1.5 cm from the base of the membrane. An unmodified DNA oligonucleotide, which is complementary to the reporter probe sequence on the liposomes, is applied to form the control zone 2.5cm from the base of the membrane. (B) In the presence of target, a sandwich hybridization complex forms with dye-encapsulating liposomes resulting in a magenta-colored band at the capture zone. (C) In the absence of target, only the control band is visible as no sandwich complex has formed.

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