The first radioimmunoassay for plasma testosterone was developed at the end of the sixties (Furuyama et al. 1970). It was based on an antiserum raised against testosterone coupled to bovine serum albumin at position 3 (T-3-BSA), 3H-labelled testosterone as the tracer and bound/free separation by ammonium sulfate precipitation. Plasma testosterone was extracted and chromatographed on alumina columns prior to immunoassay. Radio- and other immunoassays for plasma and serum testosterone were developed by several investigators e.g. Nieschlag and Loriaux (1972). These authors produced their own antiserum by a novel immunization technique (Vaitukaitis et al. 1971) and distributed the antiserum freely to other laboratories so that their method became widely used and their papers were ranked as Citation Classics in 1981. Slowly, kit manufacturers took over the development of assays, but while the practicability of the assays consistently improved, the overall performance of current immunoassays is not much different from that of 30 years ago. For instance, the early assays already showed a lower detection limit of 3-10 pg, as the current assays do. Since RIAs are most sensitive at low antibody concentrations, when competition between tracer and unknown is high, high affinity antibodies are crucial for sensitive assays. However, usually high affinity is better obtained with polyclonal antisera which display elevated cross-reactivity with DHT. A substantial improvement of the sensitivity of testosterone RIAs (and other immunoassays as well) could be achieved by using highly specific monoclonal antibodies with high affinity, a goal very difficult to reach.

In general the current in-house methods for testosterone RIA are the same as the early assays of the seventies and are still in use mainly for research purposes because they are cheap and accurate. An extraction step is necessary to eliminate serum proteins which do not allow the correct interaction of albumin and SHBG-bound testosterone with the antiserum. Due to the extraction, traditional RIAs are somewhat cumbersome and have been almost completely replaced by non-extraction methods for clinical use.

Most of the current commercially available RIAs for testosterone do not usually require an extraction step and are either based on double antibody separation or are in solid phase, i.e. the antibodyisfixed at the wall of the reaction tubes (coated tubes) so that no centrifugation is required, reducing the hands-on time and improving practicability. Serum testosterone is displaced from carrier proteins by chemical agents competing for protein binding, e.g. danazol (Pugeat et al. 1981), although the exact nature of the kit components is usually known only to the manufacturers and is protected by property rights. Well-validated non-extraction methods may workwell for male serum samples, although inaccurate testosterone concentrations are occasionally measured in individual samples containing abnormal SHBG concentrations orsubstances (e.g. drugs) interfering with the kit components. However, results obtained with different RIA kits have been repeatedly reported to be poorly comparable (Jockenhovel etal. 1992; Boots etal. 1998;Taieb etal. 2003; Wang etal. 2004).

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