Choice of label

5.2 Choice of label

When choosing a ‘label’ for use as means of detection within an immunoassay, the following should be considered: • the nature of the label; • the characteristics of signal generation; and • the detection system used to measure it.

Radioisotopes and enzymes have so far found wide application in immunoassay. The type of label can have a marked effect on the maximum sensitivity of the assay system.

Please refer to the following websites for comprehensive details on radioisotope health and safety procedures as well as useful information regarding detection and half-life: http://www.practicingsafescience.org http://www.hse.gov.uk

5.2CHOICE OF LABEL

5.2.1 Radiolabels

Two main factors contribute to the accuracy and sensitivity of signal detection: (i) the level of background; and (ii) the number of measurable units of activity generated per unit time for each labelled molecule. Sensitivity will be at its greatest when the background is near zero and each molecule of bound antibody emits an observable signal.

I has been frequently used as it has the advantages of: (i) a low natural background in the environment and in biological fluids; and (ii) a radioactive disintegration that is independent of the chemical or physical nature of the

The radioisotope 125

assay. The main problem with this seemingly useful isotope is a handicap of about six orders of mag-

nitude on assay sensitivity; e.g. under normal conditions, the background count in a radioassay is unlikely to be less than 30 c.p.m. Assuming a detection efficiency of 50% of radioactive disintegra- tions, if each reagent molecule is labelled with a single 125

I molecule, we will need about 250 000 molecules of antibody to obtain a count rate of 1 c.p.m. To get a statistically significant result compared to background, many millions of antibody molecules need to be bound, rather than the theoretical threshold for immunometric assays of a single molecule. The half-life of the chosen isotope must be considered since an isotope with a short half-life will have a limited shelf life.

5.2.2 Enzyme labels

Enzymes offer a distinct advantage over radioisotopes in that each enzyme-labelled antibody can contribute to the signal during the time of the assay, as each enzyme molecule can convert many molecules of substrate to detectable product. Most enzyme assays are limited by the sensitivity of the detection apparatus (photometer, spectrophotometer) and the poor optical and chemical reproducibility of the cuvettes, microtitre trays, etc., used for the assays. The strategy adopted by Harris et al. (1979) was to use an enzyme label (alkaline phosphatase) to convert a radioisotop-

ically labelled substrate ( 3 H-adenosine monophosphate), rather than a chromogenic substrate, into a labelled product ( 3 H-adenosine). This procedure permits the sensitivity of radioactive detection to be combined with enzyme amplification, thus giving a detection sensitivity of 10 –21 moles (about 600 molecules), much closer to the theoretical threshold.

In competition immunoassay, as the threshold of detection is high and depends upon anti- body affinity rather than on the ability to detect single molecules of reagent, increases in specific activity only contribute to increased assay speed. Thus conventional radioisotopic methods are still the method of choice in many of these assays.

For details of biotinylation and its use in assays, see Section 4.7.

5.2.3 Methods for detection of label

The method depends on the label; e.g. 125

I is easily detected in a γ spectrometer scintillation

counter and many systems are now available for counting multiple samples at the same time.

Detection by liquid scintillation, e.g. for 3 H, can be a very sensitive method, but there is the problem of quenching in that some biological molecules within the reagents may quench the scintillant (see also Stokke et al. 1990).

C H A P T E R 5: Immunoassay

Enzymes are generally used to produce coloured products from colourless substrates that can

be determined easily in a spectrophotometer or colorimeter. Automated plate readers are com- mercially available which make reading large numbers of samples relatively easy.