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Immunoassay of Theophylline by fluorescence polarisation

zation that is totally randomized.



The degree of polarization is defined as

where Pr is defined as I||/ I, with I|| the intensity of emitted light polarized in the same direction as the excitation beam, and I the intensity of emitted light polarized perpendicular to the excitation beam. In practice, I|| = IVV, where V is the polarization vector normal to the optical plane, and the double subscript refers to the orientation of excitation and emission polarizers (see Figure 1). I should be equivalent to IHH, IHV, and IVH, where H is the polarization vector lying in the optical plane. Because of instrument artifacts, however, to IHH, IHV, and IVH are not equivalent. Measuring all four intensities is therefore necessary. The artifacts are then excluded by calculating PP:

The required number of measurements may be reduced to two by observing the emission simultaneously through two polarizers, using two detectors (Figure 2). In this T-format, one emission polarizer is fixed in the H position, the other in the V position, while the excitation polarizer is rotated between the V and H positions. With the SPEX Third Polarization Unit, the emission wavelength of one detector is selected by the emission monochromator, while the other detector has its wavelength selected by a filter.

Though convenient and economical, the filter method is insufficient when highly scattering samples are being analyzed, or scanning the emission spectrum is necessary, or when two wavelengths must be monitored simultaneously. In such cases, the T-format Sample Module must be employed, for it mounts two emission monochromators at 90 to the excitatio
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