Essential Endocrinology and Diabetes. Richard I. G. Holt

Figure 4.1 The basics of immunoassay are shown for growth hormone (GH; see text for details). For clarity, in Figures only small numbers of hormone molecules and antibodies are shown; in practice, numbers are in the order of 10⁸–10¹³.

       Immunoassays – the competitive‐binding assays

In the competitive‐binding immunoassay (shown for T₄ in Figure 4.3), constant amounts of antibody and labelled antigen are added to each tube. A ‘zero’ tube is set up that contains labelled T₄, as well as a tube that also includes a known amount of unlabelled standard T₄. Incubation allows the antigen–antibody complex to form. Since the zero tube contains twice as much labelled T₄ as antibody, half of the labelled hormone will be bound and the other half will remain free (i.e. in excess). In the other tube, unlabelled and labelled T₄ compete for the limited opportunity to bind antibody. The total antibody‐bound T₄ is separated (e.g. by precipitation) and the label measured (e.g. by fluorescence or radioactivity). There will be less signal from the second tube because of competition from the unlabelled T₄; the decrease will be a function of the amount of unlabelled T₄ added, i.e. the signal decreases as the amount of unlabelled T₄ increases, allowing the construction of a calibration curve (Figure 4.3). For clinical use, standard T₄ is replaced by the patient sample, with all other assay conditions kept the same. As for immunometric assays, a five to eight‐point calibration curve offers sufficient precision for patient samples to be interpolated.

Figure 4.2 The basics of an immunometric assay for growth hormone (GH; also see text). As in Figure 4.1, in practice, large numbers of molecules are present for each reagent and the incubation of the first and second antibodies is usually simultaneous. Because the hormone is bound between the two antibodies in the triple complex ( ), this assay is sometimes referred to as a sandwich assay. Separation of the complex from the excess labelled second antibody is usually achieved physically, e.g. by precipitation and centrifugation (the supernatant contains the unbound antibody and is discarded). This leaves the bound labelled second antibody to be quantified by counting radioactivity or measuring fluorescence. The low measurement from the ‘zero’ tube, Tube 1, and the higher value from Tube 2 are plotted on the calibration curve. Tube 1 is not zero because of minor non‐specific antibody binding. The calibration line is also curved, rather than straight, because of the reversible nature of the interaction between antibodies and their antigens. In practice, five to eight calibration points are used to construct the curve.

      Analytical methods linked to mass spectrometry

In some situations, immunoassays are unreliable or unavailable, commonly because antibodies lack sufficient specificity, or there are difficulties with measurements at low concentrations (e.g. serum testosterone in women). This leads to differences in measurements across different assay platforms that inhibit the development of internationally agreed standards for diagnosis and care. For some steroid or peptide hormones, or metabolic intermediaries, mass spectrometry (MS) is becoming increasing helpful. It is applied either by itself or, for increased ability to resolve and measure substances, in tandem (MS/MS) or downstream of liquid chromatography (LC/MS) or gas chromatography (GC/MS). These approaches provide definitive identification of the relevant hormone or compound according to its chemical and physical characteristics, e.g. particularly useful for the unequivocal detection of performance‐enhancing agents in sport.

Figure 4.3 The basics of an immunoassay for thyroxine (T4; also see text). As in Figure 4.1, in practice, large numbers of molecules are present for each reagent. Under the conditions shown, the competition between equal amounts of labelled and unlabelled T₄ in Tube 2 will be such that, on average, 50% of the antibody binding sites will be occupied by labelled T₄. Because of competition between labelled and unlabelled hormone for a limited amount of antibody, this type of immunoassay is sometimes called a ‘competitive‐binding’ assay. After removing unbound label (as in Figure 4.2 legend), the fluorescent or radioactive bound fraction is quantified and a calibration curve constructed. In practice, five to eight calibration points are used to construct the curve.

      GC allows separation of vaporized molecules according to their chemical structure. For a sample loaded on a GC column, different components exit the column and pass to the mass spectrometer at different times. MS ionizes compounds to charge them, after which the spectrometer measures mass and charge during passage through an electromagnetic field. This gives a characteristic mass‐to‐charge ratio for any one substance. As with immunoassays, patient samples can be judged against the performance of precisely known standards. LC/MS is similar to GC/MS; however, the initial separation is performed in the liquid rather than the gaseous phase.

      Enzymatic assays

      Some metabolites are assayed enzymatically, frequently using dye substrates that are catalyzed to products that are coloured or fluoresce. By incorporating known standards, the amount of colour or fluorescence can be used for precise quantification. For example, glycated haemoglobin (HbA_1c), a measure of long‐term diabetes control (Chapter 11) can be measured in an enzymatic assay as well as by immunoassay and chromatography/MS approaches. Serum glucose can be measured by oxidation to generate a product that interacts with a dye to generate colour or fluorescence in an enzymatic assay.

Reference ranges

Typical adult reference ranges are listed for a number of hormones in Table 4.1. Whenever possible, hormones are measured in molar units (e.g. pmol/L) or mass units (e.g. ng/L). However, this is not possible for complex hormones such as the glycoproteins thyroid‐stimulating hormone (TSH), luteinizing hormone (LH) and follicle‐stimulating hormone (FSH), because they circulate in a variety of slightly different forms (‘microheterogeneity’). In this scenario, international reference preparations are agreed, with potency expressed in ‘units’ (U) and their subdivisions [e.g. milliunits (mU)]. Potency is assigned after large collaborative trials involving many laboratories worldwide using a range of assay platforms and physical analytical techniques. Patient results are then expressed relative to the reference data.

Static and dynamic testing

Most endocrinology testing is ‘static’; hormones and metabolites are measured as they circulate at any one time. However, rhythmical, pulsatile