Automated fluorescence polarization

B Rollman, et al. Dibekacin assay in serum by automated fluorescence polarization immunoassay (Abbot TDX)—Comparison with high performance liquid chromatography, substrate labeled fluorescent immunoassay and radioimmunoassay. J Pharm Biomed Anal 4 53, 1986. 

The fluorescence polarization immunoassay is used for routine, automated immunoassay of small molecules, such as drugs. It depends on the principle that a fluorophore attached to a macromolecule such as an antibody is not free to rotate in solution. If polarized light is used to stimulate the fluorophore to fluoresce, emission from the bound fluorophore (attached to the antibody, which is bound to a surface) will continue to be polarized, but polarization will be lost from free fluorophore (111). 

JoUey ME, Stroupe SD, Schwenzer KS, et al. Fluorescence polarization immunoassay. III. An automated system for therapeutic drug determination. Clin Chem 1981 27 1575-9. 

Nonisotopic methods have also been described. For example, a homogeneous (nonseparation) fluorescence polarization immunoassay for DHEA-S that uses a rabbit polyclonal antibody and a DHEA-fluorescein tracer is available. The measured polarization is inversely related to DHEA-S concentration. This fully automated system has a dynamic range of 1 to lOOOjJ-g/dL (0,03 to 27 Limoi/L), and interassay coefficients of variation are less than 10% over a broad concentration interval (25 to lOOOpg/dL 0.7 to 27pmol/L). Assay time is about 15 minutes for a single sample and 30 minutes for 20 samples. 

Vukjovic et al.199 recently proposed a simple, fast, sensitive, and low-cost procedure based on solid phase spectrophotometric (SPS) and multicomponent analysis by multiple linear regression (MA) to determine traces of heavy metals in pharmaceuticals. Other spectroscopic techniques employed for high-throughput pharmaceutical analysis include laser-induced breakdown spectroscopy (LIBS),200 201 fluorescence spectroscopy,202 204 diffusive reflectance spectroscopy,205 laser-based nephelometry,206 automated polarized light microscopy,207 and laser diffraction and image analysis.208... 

An interesting detection system which needs no separation step and is amenable to automation is the Abbott TDx analyzer. It is based on measurement of increase in polarization of the fluorescence of a small fluorescent labeled antigen when bound by a larger molecular weight antibody. It has been used for detection of contaminants, especially smaller antigens like drugs and steroids in food (76). Its detection range is pg/ml which can be... 

Liquid chromatography has proven to be a valuable instrumental analytical tool for toxins owing to its capability to analyze polar, nonvolatile compounds. It is easily automated and provides excellent quantitative precision. However, problems in applying the technique exist— above all the lack, for most toxins, of analytical standards as weU as of a chromophore for sensitive UV or fluorescence detection. 

The instrumentation of HPCE is uncomplicated (see the schematic drawing in Figure 1). Briefly, both ends of the narrow-bore fused silica capillary are immersed into reservoirs containing a buffer solution that also fills the capillary. The reservoirs also contain electrodes that provide electrical contact between the high-voltage power supply and the capillary. The sample is loaded onto the capillary by replacing one of the buffer reservoirs by a sample reservoir and applying external pressure (hydrodynamic injection) or an electric field (electrokinetic injection). After the injection, the reservoir is replaced, the electrical field is applied, and the separation starts. The detection is usually performed at the opposite end of the capillary (normal polarity mode). UV/vis detection is by far the most common detection technique in HPCE. Other techniques include fluorescence, amperometry, conductivity, and mass spectrometry. Modem HPCE instruments are fully automated and thereby allow easy operations and precise quantitative analyses. 

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