Sandwich Hybridisation

In the classical hybridisation techniques, the nucleic acid target was usually fixed on a solid phase (nitrocellulose, nylon membrane, glass slide) and detected by a labelled probe added in the hybridisation buffer to the solid phase. These techniques, such as dot blots, colony blots, Southern blots, Northern blots and in situ hybridisation, usually either lacked sufficient sensitivity due to background hybridisation or were too laborious for the routine setting. The sandwich hybridisation (Figure 1) technique turned out, however, to have sufficiently low background and strong enough signal to be employed for some routine laboratory purposes. In this technique, the capture probe is fixed to the solid phase and available to the target in the hybridisation buffer. Following annealing between the capture probe and the specific target, unbound material is removed by washing and a new hybridisation buffer containing a detection probe is added.
The detection probe is typically labelled with either biotin or an enzyme and is designed to bind to a different region of the target nucleic acid than the capture probe. The detection probe will therefore stick to the capture probe and the solid phase only if bridged by the specific target. Following a wash, retained detection probe can be visualised by addition of a colourless substrate that will change to a coloured product by the enzyme conjugated to the detection probe or by a streptavidin–enzyme complex in the case of a biotin-labelled detection probe. The solid phase may be a magnetic particle or the bottom of a 96-well plate to facilitate automation. The sandwich hybridisation technique is used in commercial tests for the detection of human papillomaviruses (HPVs) in samples from the female cervix (Digene) and in the visualisation step of conventional PCR assays (e.g. several Roche assays). The principle is also utilised in line probe assays.

Figure 1: Principle of sandwich hybridisation