such as viruses, and other substances in blood. The antigen is the substance or agent to
be measured. In this technique the antigen is immobilised on to a solid phase, either
the reaction vessel or a bead. The most commonly used solid phase is the enzyme
linked immunosorbent assay (ELISA) plate. Immobilisation is achieved by the use of a
coating antibodywhich actively traps antigen to the solid phase. A second antibody
(antibody enzyme conjugate) which is labelled with areporter enzymeis allowed to
bind to the immobilised antigen. Theenzyme substrateis then added to the antigen/
antibody/enzyme complex and a reaction, usually involving a colour change, is seen
(Fig. 7.12, see also colour section). There are many permutations of this method but
all of them rely on the antibody–antigen complex being formed and the presence of it
being confirmed by the reactions of the reporter enzyme. These assays rely on a
stepwise addition of layers with each one being linked to the one before. The antigen
is central to the assay as it provides the bridge between the solid phase and the signal-
generating molecule. Without antigen, the antibody enzyme conjugate cannot be
bound to the solid phase and no signal can be generated. The coating antibody also
concentrates the antigen from the sample as it binds the antigen irreversibly and so
the coating layer has the ability to concentrate available antigen until saturation has
been reached. This is particularly useful when testing for low levels of antigens in
fluids such as blood serum.Fig. 7.12A microtitre plate. (See also colour plate.)286 Immunochemical techniques