Detection, signal processing, and calibration In lmmunoassay systems

The new trends in immunochemistry related to the replacement of radioisotopic labels with non-radioactive labels are presented. Immunoenzymatic, fluorescent and chemiluminescent techniques are described in terms of their basic principles and their most common applications. The advantages of computer-controlled calibration are also discussed.


Introduction
The problems of signal detection and elaboration in nonisotopic assays are clearly related to the kind of label used. Various physical properties (other than radioactivity) of the label itself, or, alternatively, of labelmediated products are measured to monitor immunochemical reactions.
The signal can be detected either continuously or in a non-continuous way. An example of the first case is the measurement of the optical density of an enzyme mediated reaction; photon counting during fluorescent or chemiluminescent reactions is typical of non-continuous detection. Radioisotopes, which have been the most popular labels for immunochemical reactions in the last 30 years, are increasingly being replaced by nonisotopic labels; nevertheless, radioisotopes are the 'gold standards' when comparing the performanceg (sensitivity and specificity) of new labels.

Specific activity and detection limits
The term 'specific activity' was used in the past to indicate the number of radioactive disintegrations per unit of time per unit of weight of the isotope compound in radioisotopic labelled immunoassays. It is now used, in a wider sense, to indicate detectable events per unit of time per unit of weight of label material in nonisotopic mediated reactions. As shown in table 1, the specific activity of chemiluminescent and fluorescent labels is notably higher than with radioisotopes ]. This does not mean that chemiluminescent of fluorescent labels always result in more sensitive tests: in fact, many factors play an important role in determining sensitivity: the most important one is the possible steric hindrance due to some  substrates for the secondary system, driven by the enzymes phosphofructokinase and fructosebisphosphatase, with production of inorganic phosphate. Redox cycle is a secondary amplifying system as shown in the right-hand side of the figure: the labelling enzyme ALP, acting as primary system enzyme, generates the substrate nicotinamide adeninnucleotide (NAD) for the redox cycle driven by the enzymes alcoholdehydrogenase and lipoamidedehydrogenase; the cycle needs an excess of ethanol and generates formazan dye as product. A ten-fold increase in absorbance can be obtained in this way [5]. Nonisotopic methods based on photon counting as signal measurement include fluorescence and chemiluminescence.

Fluorescent and chemiluminescent techniques
In fluorescence a molecule (fluorophor), excited by light, moves to an excited state and then returns to the natural state. Energy is emitted during this process as photons and then measured without chemical modification.
Conversely, chemiluminescence is based on a chemical transformation of a substance into another one. In such a chemical reaction, energy is emitted and then measured.
In the field of fluorescent immunoassays (FIA), various methods have been implemented and are routinely used; the most popular are described below.
Fluorescence polarization immunoassay (FPIA) is characterized by a fluorogenic label being excited by polarized light and emitting a partially polarized fluorescence at a right angle to the incident beam.
Front surface fluorescence is characterized by a fluorescence mediated immunoreaction taking place on a solid-phase device, reflecting the generated fluorescence in the same direction as the exciting light.
In time resolved fluorescent immunoassays (TRF) immunoreagents are labelled with a special class of fluorogens (lanthanide ions, usually Europium), characterized by emission peaks at 614 nm, very narrow and well separated from the scattering caused by excitation (at 340 nm) and from interfering fluorescence of serum (from 400 to 600 nm) (see figure 2). In addition, the specificity of this technology is further enhanced by the decay time of these substances, which is much longer (10-1000 s) than that of the background (1-20 ns); thus, specific fluorescence can be detected once the interfering one has fully decayed [6].
Luminescence is based on the property of some natural (bioluminescence) or non-natural (chemiluminescence) chemical compounds to emit energy (photons) when Table 4. Stopping solutions currently used in immunochemistry. chemiluminescence--this results in a raised intensity of the emitted light, as well as in its transformation from a flash to a constant glow. A 1000 times amplification and a stabilization up to 20 minutes of the signal is obtained in this way, thus allowing an optimization of the reaction [7].

Calibration
Calibration is an important step in an immunochemical reaction: it is classically performed by using various standards, containing different, well-defined amounts of the analyte. This calibration, typical of the manual procedures, has been applied to semi-automated and automated instruments. The introduction of automation to immunochemistry has meant that the calibration points in the daily work have been reduced to one or two points, while the complete multipoint standard curve is stored in the computer. The advantages of such procedure are obvious.
A very simple and inexpensive calibration system is represented by so-called 'master curve': the 'true' calibration in these cases is performed by the manufacturer and memorized by the computer which is then able to retrieve the calibration data following an instruction from the operator (usually represented by a barcode connected to a single lot of reagents).