6. Wash

Figure 7.12 The two-site assay employing two monoclonal antibodies directed against two distinct epitopes.

7. Incubate with enzyme substrate and measure product e

> Enzymatic cycling -see Section 8.5.1.

> Fluorescence Section 2.4.2.

see to remove the remaining biological material a second, enzyme-labelled, monoclonal antibody is added which recognizes a different epitope on the antigen (the second site), thus sandwiching the antigen between the enzyme-labelled and the solid-phase antibodies. The excess enzyme-antibody is washed away before addition of a suitable substrate and development of a colour which is measured either kinetically or as an end-point reaction.

The enzymes commonly used as labels include alkaline phosphatase (EC, horseradish peroxidase (EC and beta-galactosidase (EC The enzymes used should be capable of being covalently linked to the antigen or antibody without loss of either catalytic activity or immuno-reactivity. Glutaraldehyde may be used as a linking agent, while glycoprotein enzymes such as peroxidase may be linked via the carbohydrate group using periodic acid to form a reactive aldehyde group.

The choice of enzyme is governed by the availability of substrate and the type of detector. Several substrates have been developed for use with horseradish peroxidase: o-phenylene diamine (OPD), 2,2-azino-di(3-ethylbenzo-thiazoline-6-sulphonate) (ABTS) and 5,5'-tetramethylbenzidine hydrochloride (TMB).

Enzymes may also be used to amplify the signal from the label, using cycling systems. Amplification of the signal from the label has the potential to increase the sensitivity of the immunoassay system.

Luminescent labels

There are several different types of luminescence differing only in the source of energy used to excite the molecules to a higher energy state; radioluminescence occurs when energy is supplied from high energy particles; in chemilumines-cence, energy is derived from a chemical reaction; in bioluminescence the excitation is performed by a biological molecule such as an enzyme; and in fluorescence or photoluminescence the excitation is derived from light energy. Each has been successfully used as a label system for immunoassay.

In chemiluminescence immunoassay the antigen is tagged with a molecule such as luminol or an acridinium ester which emits light with a high quantum yield on oxidation. Alternatively, the antigen may be labelled with a bio-luminescent molecule such as luciferin, which emits light when oxidized by the enzyme luciferase.

Successful immunoassays have been developed using photoluminescent labels such as fluorescein and rhodamine but there are significant drawbacks to the use of these compounds. A background signal can be generated by compounds present in the sample which themselves fluoresce when excited by light of the same wavelength as the fluorophore employed as the label. In addition, many biological molecules will act to decrease the emitted light by absorbing the energy, a process known as quenching. Biological molecules can also scatter the exciting light energy in such a way as to reduce the efficiency of excitation. Each of these effects has contributed to the limited usefulness of fluorescent labels in immunoassay.

The problem of background fluorescence has been largely overcome with the introduction of time-resolved fluorimetry. This technique relies on the use of fluorophores with a long-lived fluorescence which can be measured after the background fluorescence has decayed. The usual decay time for background fluorescence is about 10 ns while that of long-lived fluorophores such as chelates of the lanthanide metal europium is of the order of 103-106 ns (Figure 7.13). Instead of continuous excitation of the fluorophore, the exciting light is pulsed and readings made after the background emissions have decayed. Once a measurement has been taken, the fluorophore is pulsed again allowing the accumulation of signal from the label. Assays based on this principle have been shown to be both as precise and as sensitive as radioisotopic assays.

1 - Background fluorescence

2 - Europium chelate fluorescence

Figure 7.13 Decay curve for europium chelates showing reading time after the decay of background fluorescence.

1 - Background fluorescence

2 - Europium chelate fluorescence

Figure 7.13 Decay curve for europium chelates showing reading time after the decay of background fluorescence.

Standard materials

The quantitation of antigen will ultimately rely on comparison of test antigen responses with those of a series of standard solutions, and the type of standard solution will depend upon the nature of the assay. The matrix of the standard solution should resemble the sample matrix (blood, urine, saliva, etc.) as closely as possible and should of course be antigen free. This may be difficult to achieve in practice and only reputable suppliers should be used.

Once the assay reagents have been optimized, a standard curve can be prepared. It is customary to express the binding of labelled antigen as a percentage of the binding of the zero standard (B/B0), although this is by no means the only way of expressing the final result. The responses of the unknown samples may now be noted on the curve and their concentrations conveniently read off (Figure 7.14).

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