Fig. 21.1 Principle of immunological testosterone assay. Testosterone in the serum sample (Ag) and labeled testosterone (Ag*) competes for binding to a limited number of binding sites (Ab). The reaction is governed by the law of mass action. The asterisk indicates any type of label (e.g. an isotope, a non-radioactive label, an enzyme, etc.). At the end of the reaction the Free is removed and the Bound is counted.

by the antibody. At the end of the reaction, the antibody bound to the hormone (B) is separated from the free fraction (F) and the radioactivity or the signal emitted by a non-radioactive tracer is measured (Fig. 21.1).

In case of testosterone RIA, the tracer can be tritiated or iodinated. 3H-testosterone can be tritiated in two or four positions (Fig. 21.2). Iodination, which can be achieved by oxidation (e.g. by reaction with chloramine-T) of a thyrosine or another amino acid residue in peptidic hormones, requires conjugation with a histamine residue in the case of a steroid molecule (Fig. 21.2). Both tritiated and iodinated testosterone tracers are commercially available. The half-life of the tracers depends on the isotope. Tritiated tracers can be stored and used for years but, since the slow but progressive decay results in impurities which reduce the assays' performance, they should be purified by chromatography at 6-12 months intervals. Iodinated tracers, e.g. testosterone-3-(O-carboxymethyl)oximino-(2-[125I] iodohistamine, show a much shorter half-life and can be used only for about one month, but they have a much higher specific activity than the tritiated tracers allowing the use of lower antiserum concentrations and improved assay sensitivity. Iodinated testosterone is usually purified by high performance liquid chromato-graphy (HPLC) by the manufacturer and does not require further cleaning before use. If the tracer is produced in-house, it should be purified by HPLC or other chromatographic technique (e.g. gel filtration on Sephadex) before use. In recent years non-radioactive testosterone tracers have been produced and are widely used in clinical routine measurements (see below). They offer the advantage of a lower environmental impact, but the assays employing such tracer function according to the same principles of RIAs.

Beside the specific activity of the tracer, the assays' sensitivity depends on the affinity of the antiserum, which should, if possible, be identical for both the antigen and the tracer. Since testosterone is not antigenic when injected in animals, a testosterone



Fig. 21.2 Tracers used in testosterone immunoassays. RIAs are based on tritiated or iodinated (testosterone-3-(0-carboxymethyl) oximino-(2-[,25I]iodohistamine) tracers. The Europiumlabeled testosterone is an example of non-radioactive tracer used in fluoroimmuno assays (FIA). Other non-radioactive immunoassays are based on testosterone molecules coupled with enzymes or luminescent substances.

Fig. 21.2 Tracers used in testosterone immunoassays. RIAs are based on tritiated or iodinated (testosterone-3-(0-carboxymethyl) oximino-(2-[,25I]iodohistamine) tracers. The Europiumlabeled testosterone is an example of non-radioactive tracer used in fluoroimmuno assays (FIA). Other non-radioactive immunoassays are based on testosterone molecules coupled with enzymes or luminescent substances.

conjugate conferring aptene properties to the steroid must be used to obtain antisera. As for iodination, position 3 in the A ring of testosterone is usually exploited for conjugation with the CMO (carboxymethyloximino) group, a spacer necessary for coupling the antigen to BSA which renders the conjugate antigenic. The antibodies for testosterone immunoassays are usually polyclonal antisera obtained in rabbits, but monoclonal antibodies are used in some kits. Polyclonal antisera have the advantage of high affinity, but good monoclonal antibodies might have better specificity, obviating, at least in part, the problem of cross-reactivity of most polyclonal antisera with DHT.

After the antigen-antibody reaction has reached equilibrium, separation of the antibody-bound (B) from the free tracer (F) can be accomplished specifically by adding an antiserum directed against the immunoglobulins of the species from which the first antibody was obtained (e.g. goat antirabbit antiserum), together with preimmune serum (e.g. normal rabbit serum) to achieve complete precipitation of the immune complexes. The reaction tubes are then centrifuged and the radioactivity or the signal emitted by the non-radioactively-labeled tracer is counted.

Alternatively, non-specific precipitating agents (e.g. ammonium sulphate, polyethylene glycol, [PEG]) or subtances which absorb the free antigen (e.g. dextran-coated charcoal) can be used. In practice, in a testosterone RIA based on rabbit antiserum, B/F separation is performed very efficiently by addition of rabbit immunoglobulins, antirabbit antiserum and PEG.

After counting the results can be calculated in several ways. The signal emitted by the unknown samples is compared to that of the samples with known testosterone concentrations, i.e. the calibrators of the standard curve, after logarithmic, semilog-arithmic or logit/log transformation, using computer programs usually enclosed in the software of the counter. These mathematical transformations of the readouts permit linearization of the calibration curve over a wide range, allowing accurate calculation of the actual testosterone concentration in the unknowns.

Since testosterone in serum is mostly bound to carrier proteins, which prevents the antibody-antigen reaction by competing with the antiserum, the steroid must be extracted with organic solvents prior to RIA or other immunoassays. Extraction is usually performed by adding 10-20 volumes of diethyl ether to the serum samples. This step is followed by vortexing (5 min) or agitation of the samples on a rotator (30 min) and freezing of the aqueous phase. Testosterone, which is lipophilic, remains in the organic phase which can be decanted, evaporated and reconstituted in assay buffer. This extraction procedure is usually highly efficient (^90%) and can be monitored by measuring the recovery of trace amounts of radiolabeled testosterone added beforehand. Both testosterone and DHT are extracted by this method. If an accurate quantification of testosterone and DHT is desired, the extracted steroids can be reconstituted in the appropriate diluent and separated by a chromatographic procedure (e.g. HPLC or celite chromatography) before RIA.

The calibrators used in the standard curve are serial dilutions of a sample with known testosterone concentrations dissolved in the same matrix (buffer or serum-based) of the samples measured. In extraction methods, testosterone is weighed, dissolved in ethanol and further diluted in assay buffer. In non-extraction methods the standard is added to steroid-free sera. The maintenance of the same matrix is necessary to ensure parallelism between standards and unknowns.

These assay principles are common to RIA and non-radioactive methods. Unlike the most recent assays for peptidic hormones, the newest technologies which have highly improved sensitivity thanks to the two-site, sandwich approach, cannot be applied to the steroid hormone assays. In the immunoradiometric assays (IRMA) the large protein hormone is first reacted with a capture antibody (in molar excess) coated to the tube walls, the tubes are washed,and a labeled second antibody directed against a second epitope of the hormone is added. Steroid hormones are too small to be reacted simultaneously with two antibodies and the IRMA principle cannot be applied, so that the sensitivity of a testosterone assay can be improved only by increasing the specific activity of the tracer and/or the affinity and specificity of the antibody.

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