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Amount of added antigen (log concentration)

Figure 7.11 Cross-reactivity. Compound B displaces 50% of the label at 100-fold greater concentration than compound A and is said to have a 1% cross-reactivity.

The label

Immunoassays require a pure sample of either antibody or antigen for labelling with an appropriate molecule. Such a molecule should retain a high signal efficiency (i.e. be readily detected at low concentration) while its incorporation into the antigen or antibody should have no effect on their subsequent immunoreactivity.

The choice of label is important and the most common axe listed in Table 7.3.

Radioisotopes

The most commonly used isotopes are carbon-14, tritium (hydrogen-3) and iodine-125. Carbon-14 and tritium are both beta-emitting isotopes while iodine-125 emits both beta particles and gamma radiation. Carbon-14 and tritium are examples of internal labels since the radioactive atom replaces an existing atom within the antigen, while iodine-125 is described as an external label because it is usually necessary to attach the iodine covalently to the antigen. There are advantages and disadvantages to each of these labels.

The beta-emitting carbon-14 and tritium have the advantage that the labelled form of the molecules is identical to the unlabelled antigen, but suffer from the disadvantage that the efficiency of the measurement of the beta emission, which uses the technique of scintillation counting, is less than that of the gamma emissions associated with iodine-125. Equally, iodine-125 suffers from the problem that the covalent attachment of the isotope to the antigen often means that there is a significant structural difference between the unlabelled and the labelled antigen. The gain in signal measurement over the potential loss of immunoreactivity, however, is sufficient to make iodine-125

> Chromatography see Section 3.?.

an isotope of choice for immunoassay systems. Iodine may be readily substituted onto the aromatic side-chain of the amino acid tyrosine by mild oxidation using a variety of agents; chloramine T, the enzyme lactoperoxidase (EC 1.11.1.7) and the sparingly soluble agent 'iodogen' (l,3,4,6-tetrachloro-3,6-diphenyl-glycouril) have all been used successfully to yield a stable and efficient label. In applications where there is no suitable tyrosine residue available, a carrier molecule containing both a phenol or imidazole group suitable for iodination and an amine group suitable for coupling to a carboxylate group on the antigen may be used.

After iodination the label is usually purified to remove damaged antigen and unreacted iodine and this may be conveniently accomplished using either gel permeation chromatography or HPLC.

Enzymes

Enzyme labels are usually associated with solid-phase antibodies in the technique known as enzyme-linked immunosorbent assay (ELISA). There are several variants of this technique employing both competitive and non-competitive systems. However it is best used in combination with two monoclonal antibodies in the 'two-site' format in which an excess of antibody is bound to a solid phase such as a test-tube or microtitre plate; the test antigen is then added and is largely sequestered by the antibody (Figure 7.12). After washing

1. Attachment of antibody to solid phase

2. Wash

3. Incubate with sample containing antigen o ^

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