9.2.2. Sandwich ELISA
In “sandwich” ELISA, a modified version of the primary antibody is adsorbed to the well first, in order to ‘capture’ the virus particles after the sample is added. The captured virus particles are then detected as before, either with the reporting enzyme directly conjugated to the detecting antibody (Fig. 4C) or with an extra incubation using an antibody-detecting protein conjugated to the reporter enzyme (Fig. 4D). The sandwich ELISA is cleaner and much more sensitive than conventional ELISA, but has a less predictable relationship between virus concentration and signal (depending on which component in the assay is limiting).
The most common reporter enzyme systems are
horseradish peroxidase (HRP) and alkaline phosphatase (AP). Both are relatively
robust enzymes that can be conjugated to the primary or secondary antibody.
They convert a colourless substrate into a coloured reaction product, such that
the absorbance at a wavelength appropriate for the specific colour produced is
proportional to the amount of enzyme
activity present in the sample, which in turn is proportional to the amount of
antibody captured by the sample, and thus also the amount of virus in the
sample. The protocols below are generic ones for conventional ELISA and
sandwich ELISA, based on the methods of Allen et al. (1986), using horseradish peroxidase as the reporter enzyme.
See also Harlow and Lane (1988) for alternatives and more extensive laboratory
protocols involving antibodies.
Fig. 4. Different types of ELISA, depending on whether the antigen1 is adsorbed directly onto the assay well (A & B) or is captured by the Fab fragment of a specific antibody4 or the full antibody5 (sandwich ELISA; C & D) and whether the detection system involves an enzyme reporter conjugated directly to the detecting antibody6 (A & C) or a reporter conjugated to a generic antibody-recognizing protein3 recognizing the detecting antibody2 (B & D). Adapted from de Miranda (2008).