Other immunoassays

The most popular alternatives to radioactive methods are immunoassays based on non-radioactively labeled tracers such as fluroimmunoassay (FIA), chemilumines-cent assay (LIA) and enzyme-linked immunosorbent assay (EIA/ELISA). They can be in liquid or in solid phase, whereby solid phase, microtitre plate-based assays are preferred due to easy handling and proneness to automation. Also in these assays it is the antigen that is labeled and competes with the endogenous testosterone for binding to the antiserum. The primary antibody can be poly- or monoclonal and is usually bound to the tube wall or to the plate well. In EIAs/ELISAs the tracer is represented by testosterone coupled to an enzyme (alkaline phosphatase, (p galactosidase, penicillinase, acetylcholinesterase or horse radish peroxidase) which starts a colori-metric reaction upon addition of the substrate at the end of the incubation time. This results in color development which is inversely proportional to the amount of unlabelled testosterone and can be read by a spectrophotometer. The sensitivity of EIA/ELISA is affected by the number of steroid molecules coupled to the enzyme and the proper molar ratio between steroid and enzyme must be carefully validated (Rassaie et al. 1992). In FIA the tracer is testosterone coupled to a molecule (e.g. Europium) which fluoresces upon stimulation. The fluorescence is measured by a fluorimeter and, again, is inversely related to the amount of cold testosterone contained in the sample. Critical in EIAs/ELISAs and FIAs are the washing steps, which should be carefully carried out in order to eliminate non-specifically bound substances which would result in poor precision and falsely elevated readouts.

Automatic multianalyzers, mainly based on non-radioactive methods are now available and widely used for the direct and quick measurement of serum testosterone. Some of these systems have been evaluated against the reference method based on mass spectrometry, showing acceptable results at least in male samples (Fitzgerald and Herold 1996; Levesque et al. 1998; Gonzales-Sagrado et al. 2000). However, inconsistency of results obtained with different methods are reported as well, and some systems seem to suffer from systematic problems, resulting in over- or underestimation of serum testosterone and/or insufficient sensitivity, especially in female samples (Taieb etal. 2002,2003; Wang etal. 2004). This is very evident when comparing the results of external quality control trials (Middle 2002). As for every other method, each laboratory should carefully validate the results obtained by the multianalyzer before it is implemented for routine testosterone measurement. In practice, however, validation is limited by the fact that most of the systems are based on a master calibration curve carried out by the manufacturer and not available to customers. Each assay then requires only one or two calibrators to adjust the master curve and parallelism tests cannot be performed. As in the non-automatic assays, differences observed between the kits can be ascribed to differences in the matrix of the calibrators and in the affinity, titer and specificity of the antibodies used.

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